Search

Marfan syndrome (MFS) is a complex connective tissue disorder characterized by considerable clinical variability. The diagnosis of MFS is based on the Ghent criteria, which require the presence of both clinical and genetic features. MFS is primarily caused by pathogenic alterations in FBN1, which encodes the fibrillin-1 protein. Fibrillin-1 comprises multiple domains rich in cysteine residues, with disulfide bonds formed between these residues. It has long been recognized that variants that alter or introduce cysteine residues damage protein function, leading to the development of MFS. In this study, we report a cysteine-introducing variant: FBN1 variant, c.6724C>T (p.[Arg2242Cys]). We have observed this variant in several individuals without MFS, challenging our previous understanding of the underlying mechanism of MFS. This finding emphasizes the importance of revisiting and reevaluating our current knowledge in light of new and unexpected observations. Moreover, our study highlights the significance of incorporating local and national data on allele frequencies, as well as employing multidisciplinary phenotyping approaches, in the classification of genetic variants. By considering a wide range of information, we can enhance the accuracy and reliability of variant classification, ultimately improving the diagnosis and management of individuals with genetic disorders like MFS., (© 2024 The Author(s). American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Background: Genetic findings influence clinical care of patients suspected of hereditary cardiac diseases. As additional knowledge arises over time, the classification of genetic variants may change. The labor cost associated with systematic manual reevaluation for reported variants is substantial. We applied an automated variant classifier for reevaluation of previous reported variants to assess how such tools may assist in manual reevaluation., Methods: Historically (2010-2022), patients (N = 2987) suspected of inherited cardiomyopathies or ion-channel disorders were screened for genetic variants in at least one of up to 114 genes. We had reported 1455 unique variants, of which 742 were among the 14 most relevant genes. In the 14-gene-group, we compared our reported classification to that of an autoclassifier and manually reevaluated variant classification of all variants. Among the remaining genes (N = 100), only variants where the autoclassifier predicted change of clinical impact, such as variant of uncertain significance to likely pathogenic or oppositely, were manually reevaluated., Results: We identified 9% (66/742) of variants with clinical impact in the 14-gene-group. Of these, 91% could have been identified solely evaluating the 120 variants where the autoclassifier had predicted a change of clinical impact. In the 100 remaining genes, a change of clinical impact was identified in 3% (22/713) after manual reevaluation., Conclusion: Using an autoclassifier reduces the workload to identify variants likely to have a change in variant class with clinical impact. Hence, we recommend using such tools to identify the variants most relevant to manually reevaluate to improve patient care., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as "maybe report" after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known., (© 2024. The Author(s).)

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., Competing Interests: JG declares research grants from Augustinus Foundation, Danish Medical Association, A. P. Møller Foundation, and PhD scholarship from Region of Southern Denmark PhD Fund and Faculty of Health Sciences University of Southern Denmark. HB declares consultancy and/or advisory board participation for NOVO Nordisk, Astra-Zeneca, Boehringer Ingelheim, Bayer, Alexion, GSK, Vifor Pharma, Otsuka, Galapagos; speakers honorarium from Astra-Zeneca, Astellas, Novartis and MSD; as well as research grants from Vifor Pharma, GSK, NOVO-foundation, Karen Elise Jensen Foundation and Augustinus Foundation. MR declares research grants from the Danish Kidney Association, Family Hede Nielsen Foundation, Aase and Ejnar Danielsens Foundation, Augustinus Foundation, Harboe Foundation and lecture honorarium from the board of Health., (© The Author(s) 2024. Published by Oxford University Press on behalf of the ERA.)

Genetic conditions are often familial, but not all relatives receive counseling from the same institution. It is therefore necessary to ensure consistency in variant interpretation, counseling practices, and clinical follow up across health care providers. Furthermore, as new possibilities for gene-specific treatments emerge and whole genome sequencing becomes more widely available, efficient data handling and knowledge sharing between clinical laboratory geneticists and medical specialists in clinical genetics are increasingly important. In Denmark, these needs have been addressed through the establishment of collaborative national networks called Genetic Expert Networks or "GENets". These networks have enhanced patient and family care significantly by bringing together groups of experts in national collaborations. This promotes coordinated clinical care, the dissemination of best clinical practices, and facilitates the exchange of new knowledge., Competing Interests: Conflicts of interest No conflicts of interest: DLL, ALF, BSC, CBA; CKL, HGK, ISP, LR, LS, MAR, MB, MKA, NDR, PAG, PET, SEB, SGL, SHH, TBH, BRD., (Copyright © 2023. Published by Elsevier Masson SAS.)

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., (© 2023 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

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., Competing Interests: Authors LJ, LH, RS, PS, KR, ML, JH, IC, and BN are employed by ARCEDI, a Danish biotech company that holds the proprietary technology for enrichment of fetal cells from maternal blood used in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jeppesen, Hatt, Singh, Schelde, Ravn, Toft, Laursen, Hedegaard, Christensen, Nicolaisen, Andreasen, Pedersen, Vogel and Lildballe.)

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., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

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., Competing Interests: MC received lecture fees from Amgen. HJ received lecture fees from Abbott Denmark, Amgen Denmark and Biosense Webster, Europe. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Calloe, Magnusson, Lildballe, Christiansen and Jensen.)

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., (Copyright © 2023 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Background: The existing risk of procedure-related miscarriage following invasive sampling for prenatal diagnosis is higher for twin pregnancies and some women are reluctant to test these typically difficultly obtained pregnancies invasively. Therefore, there is a need for noninvasive testing options that can test twin pregnancies at an early gestational age and ideally test the twins individually. Case presentation: A pregnant woman opted for cell-based NIPT at GA 10 + 5. As cell-based NIPT is not established for use in twins, the test was provided in a research setting only, when an ultrasound scan showed that she carried dichorionic twins. Materials and Methods: Fifty mL of peripheral blood was sampled, and circulating fetal cells were enriched and isolated. Individual cells were subject to whole-genome amplification and STR analysis. Three fetal cells were analyzed by chromosomal microarray (aCGH). Results: We identified 20 fetal cells all sharing the same genetic profile, which increased the likelihood of monozygotic twins. aCGH of three fetal cells showed the presence of two X chromosomes and a gain of chromosome Y. CVS from both placentae confirmed the sex chromosomal anomaly, 47,XXY and that both fetuses were affected. Conclusion: NIPT options can provide valuable genetic information to twin pregnancies that help the couples in their decision-making on prenatal testing. Little has been published about the use of cell-based NIPT in twin pregnancies, but the method may offer the possibility to obtain individual cell-based NIPT results in dizygotic twins., Competing Interests: LJ, LH, RS, and PS were employed by the Danish biotech company ARCEDI Biotech that has launched a commercial cell-based noninvasive prenatal test (www.evitatest.com). JH was employed by Aagaard Fertility Clinic. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jeppesen, Hjortshøj, Hindkjær, Hatt, Petersen, Singh, Schelde, Andreasen, Christensen, Lildballe and Vogel.)

Monogenic causes of chronic kidney disease (CKD) are more prevalent in adults than previously thought, as causative gene variants are found in almost 10% of unselected patients with CKD. Even so, genetic testing in patients with adult-onset CKD is uncommon in clinical practice and the optimal criteria for patient selection remain unclear. A family history of kidney disease emerges as one marker associated with a high diagnostic yield of genetic testing. We present 3 cases of adult-onset CKD with underlying monogenic causes exemplifying different modes of inheritance. Case 1 is a 60-year-old male with slowly progressive CKD initially ascribed to hypertension and diabetes despite a family history with several affected first-degree relatives. A pathogenic MUC1 variant was found, and thus we identified the first Danish family of MUC1-associated autosomal dominant tubulointerstitial kidney disease. Case 2 is a 40-year-old female with nephrocalcinosis, nephrolithiasis, and unexplainable hypercalcemia consistent with vitamin D intoxication. The family history indicated autosomal recessive inheritance, and genetic testing revealed 2 pathogenic CYP24A1 variants in compound heterozygous form associated with idiopathic infantile hypercalcemia. Case 3 is a 50-year-old male with microscopic hematuria, proteinuria, and hearing loss. Electron microscopy of renal biopsy showed thin basal membrane syndrome, and the family history indicated X-linked inheritance. A novel missense variant in COL4A5 was identified, suggesting an atypical late-onset form of X-linked Alport syndrome. This case series illustrates the heterogeneous presentations of monogenic kidney disease in adults and emphasizes the importance of family history for initiating genetic testing to identify underlying monogenic causation. Moreover, we discuss the potential impact of genetic diagnostics on patient management and genetic family counseling., (© 2021 S. Karger AG, Basel.)

Background: Cell-free NIPT and cell-based NIPT are risk-free testing options using maternal blood samples to screen for fetal aneuploidies, but the methods differ. For cell-free NIPT, the fetal fraction of cell-free DNA in plasma is analyzed with a high background of maternal DNA. In contrast, for cell-based NIPT, a limited number of the rare, intact fetal cells are isolated for the genetic analysis. This case demonstrates the differences regarding testing for fetal sex-chromosomes anomalies (SCAs) between these two tests. Materials and Methods: A pregnant woman with mosaicism for Turner syndrome opted for NIPT in first trimester. For the cell-free NIPT analysis, DNA extraction, genome-wide massive parallel sequencing, and data analysis were carried out as described by the kit manufacturer (Illumina©, San Diego, CA, USA). For cell-based NIPT, the first sample gave no result, but the woman consented to repeat cell-based NIPT. After whole genome amplification and STR analysis, fetal DNA from three individual fetal cells was subjected to chromosomal microarray (aCGH, Agilent oligoarray, 180 kb). Results: Fetal fraction was 7%, and cell-free NIPT showed 2 copies of chromosomes 13, 18, and 21 and a decreased proportion of chromosome X, suggestive of fetal Turner syndrome. In contrast, the cell-based NIPT result showed no aneuploidy and two X-chromosomes in the fetus. Conclusion: cell-based NIPT may provide a non-invasive testing option to screen for SCAs in women with mosaicism for monosomy-X in blood, where cell-free NIPT cannot discriminate whether the X-loss is maternal or fetal., Competing Interests: LJ, LH, RS, and PS are all employed by the Danish biotech company ARCEDI Biotech that has launched a commercial cell-based non-invasive prenatal test (www.evitatest.com). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jeppesen, Hatt, Singh, Schelde, Andreasen, Markholt, Lildballe and Vogel.)

Xia-Gibbs syndrome (XGS) is a neurodevelopmental disorder characterized by intellectual disability, developmental delay, seizures, hypotonia, obstructive sleep apnoea and mild facial dysmorphism. Heterozygosity for loss-of-function variants in AHDC1, encoding the AT-hook DNA binding motif containing protein 1, were discovered in 2014 as the likely genetic cause of Xia-Gibbs syndrome. We present five patients with Xia-Gibbs syndrome caused by previously unreported variants in AHDC1. Two of the patients share a frameshift variant: c.2849del (p.(Pro950Argfs*192)) in AHDC1. Despite sharing this variant, the two patients show remarkable phenotypic differences underscoring the clinical heterogeneity of Xia-Gibbs syndrome. In addition, we present a case of Xia-Gibbs syndrome caused by mosaicism for an AHDC1 variant., (Copyright © 2021. Published by Elsevier Masson SAS.)

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., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Introduction: In Denmark, non-invasive prenatal testing (NIPT) has been used since 2013. We aimed to evaluate the early clinical use of NIPT in Danish public and private healthcare settings before NIPT became an integrated part of the national guidelines on prenatal screening and diagnosis in 2017., Material and Methods: NIPT data were collected between March 2013 and June 2017 from national public registries and private providers. Results from follow-up samples (chorionic villi, amniotic fluid, postnatal blood or fetal tissue) were included from The Danish Cytogenetics Central Registry and indications and outcome from The Danish Fetal Medicine Database., Results: A total of 3936 NIPT results were included in the study from public hospitals (n = 3463, 88.0%) and private clinics (n = 473, 12.0%). The total number of prenatal tests was 19 713 during the study period: 20% were NIPT analyses (n = 3936) and 80% invasive procedures (n = 15 777). Twenty-five percent of NIPTs in the private clinics were performed before gestational week 11 +0 , whereas NIPT in public settings was used only after combined first trimester screening (P < .001). Regardless of indication, the national public sensitivity was 96.9% (95% CI 82.0%-99.8%) for trisomy 21, 100% (95% CI 46.3%-100%) for trisomy 18, 100% (95% CI 5.5%-100%) for trisomy 13, and 87.0% (95% CI 74.5%-92.4%) for any fetal chromosomal aberration. Forty-seven true-positive NIPT results included cases of common aneuplodies (trisomy 21, n = 31; trisomy 18, n = 5; and trisomy 13, n = 1), sex chromosomal aberrations (n = 7) and atypical chromosomal aberrations (n = 3). One false-negative NIPT result occurred (trisomy 21). Of 47 cases, 21 (45%) cases with a true-positive NIPT result resulted in live births by choice; 11 of these children had Down and 4 had Edwards syndrome., Conclusions: The total number of NIPT analyses was low compared with the number of invasive procedures in the implementation period. In contrast to the generally high termination rate after a positive result following invasive testing in Denmark, a high proportion of true-positive NIPT results from the public setting resulted in live births. NIPT may be an important risk-free alternative to invasive testing for a minority of women in the public setting who wish to use prenatal genetic testing for information only and not for reproductive decision-making., (© 2021 Nordic Federation of Societies of Obstetrics and Gynecology (NFOG). Published by John Wiley & Sons Ltd.)

Background: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive growth of cysts and a decline of renal function. The clinical feasibility of the number of potential disease-modifying drugs is limited by systemic adverse effects. We hypothesize that megalin, a multiligand endocytic receptor expressed in the proximal tubule, may be used to facilitate drug uptake into cysts, thereby allowing for greater efficacy and fewer side effects., Methods: The cyst expression of various tubular markers, including megalin and aquaporin 2 (AQP2), was analysed by immunohistochemistry (IHC) of kidney sections from the ADPKD mouse model ( PKD1 RC/RC ) at different post-natal ages. The endocytic function of megalin in cysts was examined by IHC of kidney tissue from mice injected with the megalin ligand aprotinin., Results: Cyst lining epithelial cells expressing megalin were observed at all ages; however, the proportion decreased with age. Concomitantly, an increasing proportion of cysts revealed expression of AQP2, partial expression of megalin and/or AQP2 or no expression of the examined markers. Endocytic uptake of aprotinin was evident in megalin-positive cysts, but only in those that remained connected to the renal tubular system., Conclusions: Megalin-expressing cysts were observed at all ages, but the proportion decreased with age, possibly due to a switch in tubular origin, a merging of cysts of different tubular origin and/or a change in the expression pattern of cyst lining cells. Megalin expressed in cysts was functional, suggesting that megalin-mediated endocytosis is a potential mechanism for drug targeting in ADPKD if initiated early in the disease., (© The Author(s) 2021. Published by Oxford University Press on behalf of ERA-EDTA.)

Objective: To compare mosaicisms in prenatal chorionic villus samples (CVSs) with corresponding postpartum placental samples., Method: We collected placentas from 15 consecutive cases of mosaicism detected in CVSs and obtained five standardized samples on each placenta after delivery. All pre- and postnatal placental samples were uncultured and analyzed by high-resolution chromosomal microarray., Results: Ten cases of mosaicism for whole chromosome aneuploidy (mWC) and five cases with mosaicism for (sub)chromosomal copy number variations (mCNVs) were included. In 5/10 mWC cases and in 4/5 mCNV cases the prenatally detected aberration was confirmed in the postpartum placenta. Three postpartum placentas revealed various complex aberrations differing from the prenatal results: (1) mosaicisms for different deletions/duplications on 9p and 9q in all samples (prenatal: mosaic 5.3 Mb duplication on 9p24), (2) different regions with deletions/duplications/loss of heterozygosity on 1p in all samples (prenatal: mosaic 2.3 Mb 1p36 duplication), and (3) mosaicism for a duplication on 5q and a deletion on 6p in one out of five samples (prenatal: mosaic trisomy 7)., Conclusion: CNVs constitute a complex subgroup in placental mosaicism. Counseling of these couples after chorionic villus sampling should not focus on the specific CNV involved, but on the nature of mosaicism and the option of amniocentesis and ultrasound., (© 2021 John Wiley & Sons Ltd.)

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common heritable kidney disease. ADPKD leads to cysts, kidney enlargement and end-stage renal disease. ADPKD is mainly caused by variants in PKD1 and PKD2, with truncating PKD1 variants causing the most severe phenotype. This study aimed to characterize variants in Danish patients referred for screening of genes related to cystic kidney disease., Methods: 147 families were analysed for variants in PKD1, PKD2 and GANAB using next generation sequencing and multiplex ligation-dependent probe amplification. If a variant was identified, relatives were analysed for the specific variant using Sanger sequencing., Results: A pathogenic or possibly pathogenic variant was identified in 87% (103/118) of patients suspected to suffer from ADPKD, according to the requisition form. In total, 112 pathogenic or possibly pathogenic variants were observed, of which 94 were unique; 74 (79%) in PKD1 and 20 (21%) in PKD2, while 41 variants were novel. No variants in GANAB were observed. Ten recurrent variants were observed in 26 (26%) families. These were either PKD2 variants (N = 6) or non-truncating PKD1 variants (N = 4). Five of these were likely founder variants., Conclusions: The distribution of pathogenic or possibly pathogenic variants in the Danish ADPKD population is similar to that in other populations, except that recurrent truncating PKD1 variants appear to be rare, i.e. founder variants tend to be variant types associated with a mild phenotype. Patients with a mild phenotype may remain undiagnosed, consequently the frequency of founder variants and prevalence of ADPKD may be underestimated., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objective: We aimed to develop cell-based NIPT for cystic fibrosis (CF) and test a pregnancy at risk of two common pathogenic variants., Method: A pregnant woman carrying monozygotic twins opted for prenatal testing as she and her partner were heterozygote carriers of F508del (c.1521:1523del). The partner was also positive for the CFTR-related variant R117H (c.350G>A). Fetal trophoblasts from maternal blood were enriched and isolated using antibodies and a capillary-based cell-picking instrument. Multiplex PCR-based fragment length analysis was performed on the extracted fetal DNA for STR-genotyping, fetal gender and F508del variant status. The R117H variant status was tested using SNaPshot analysis., Results: The fetal origin of the isolated cells was verified by detection of two paternally inherited STR alleles and an Y chromosome marker, while no maternal DNA contamination was detected. The direct variant analysis detected F508del heterozygosity and the SNaPshot analysis for R117H detected only the normal allele. Thus, the results showed that the fetuses were healthy carriers of F508del, concordant with the findings of conventional prenatal testing., Conclusion: Cell-based NIPT could accurately state the fetal variant status and distinguish fetal trophoblasts from maternal cells. In the future, cell-based NIPT may provide an accurate less invasive alternative to chorionic villous sampling., (© 2020 John Wiley & Sons Ltd.)

This review describes clinical characteristics, mode of inheritance, and molecular genetic testing for the following monogenic kidney diseases: polycystic kidney disease, Alport syndrome, autosomal dominant tubulointerstitial kidney disease, and nephronophthisis. The same is described for steroid resistant nephrotic syndrome, kidney stones and congenital anomalies of the kidney and urinary tract, which in some cases have a monogenic cause. Knowledge of possibilities within molecular genetic testing may help more kidney disease patients to receive a specific diagnosis.

Objective: Placental mosaicism for a subset of a chromosome, a structural chromosomal aberration, is thought to be a very rare finding in chorionic villus samples. Here, we present clinical and laboratory data on five cases with such mosaicism for structural chromosomal aberrations., Methods: During a period of 6 months, chromosomal microarray was carried out on DNA extracted from 100 uncultured chorion villous samples from high-risk pregnancies., Results: In five of 100 consecutively collected samples (5/100), mosaicism for a structural chromosomal aberration was detected. The mosaic aberration was subsequently detected in fetal tissue in three of the five cases., Conclusion: Chromosomal microarray can detect placental mosaicism for structural chromosomal aberrations. This kind of mosaicism may be more frequent than previously anticipated, and the fetal involvement seems difficult to predict. These findings highlight the complexity of mosaicism for structural chromosomal aberrations in prenatal samples in the chromosomal microarray era., Competing Interests: Declaration of competing interest No conflict of interest to be declared for any of the authors., (Copyright © 2019. Published by Elsevier Masson SAS.)

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., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Introduction: We present a case of an infertile male with 46,XX/46,XYchimerism fathering a child after ICSI procedure., Methods: Conventional cytogenetic analysis on chromosomes, derived from lymphocytes, using standard Q-banding procedures with a 450-550-band resolution and short-tandem-repeat analysis of 14 loci., Results: Analysis of 20 metaphases from lymphocytes indicated that the proband was a karyotypic mosaic with an almost equal distribution between male and female cell lines. In total, 12 of 20 (60%) metaphases exhibited a normal female karyotype 46,XX, while 8 of 20 (40%) metaphases demonstrated a normal male karyotype 46,XY. No structural chromosomal abnormalities were present. Out of 14 STR loci, two loci (D18S51 and D21S11) showed four different alleles in peripheral blood, buccal mucosal cells, conjunctival mucosal cells, and seminal fluid. In three loci (D2S1338, D7S820, and vWA), three alleles were detected with quantitative differences that indicated presence of four alleles. In DNA extracted from washed semen, four alleles were detected in one locus, and three alleles were detected in three loci. This pattern is consistent with tetragametic chimerism. There were no quantitative significant differences in peak heights between maternal and paternal alleles. STR-analysis on DNA from the son confirmed paternity., Conclusion: We report a unique case with 46,XX/46,XY chimerism confirmed to be tetragametic, demonstrated in several tissues, with male phenotype and no genital ambiguity with oligospermia fathering a healthy child after IVF with ICSI procedure.

Identification of fetal kidney anomalies invites questions about underlying causes and recurrence risk in future pregnancies. We therefore investigated the diagnostic yield of next-generation sequencing in fetuses with bilateral kidney anomalies and the correlation between disrupted genes and fetal phenotypes. Fetuses with bilateral kidney anomalies were screened using an in-house-designed kidney-gene panel. In families where candidate variants were not identified, whole-exome sequencing was performed. Genes uncovered by this analysis were added to our kidney panel. We identified likely deleterious variants in 11 of 56 (20%) families. The kidney-gene analysis revealed likely deleterious variants in known kidney developmental genes in 6 fetuses and TMEM67 variants in 2 unrelated fetuses. Kidney histology was similar in the latter 2 fetuses-presenting a distinct prenatal form of nephronophthisis. Exome sequencing identified ROBO1 variants in one family and a GREB1L variant in another family. GREB1L and ROBO1 were added to our kidney-gene panel and additional variants were identified. Next-generation sequencing substantially contributes to identifying causes of fetal kidney anomalies. Genetic causes may be supported by histological examination of the kidneys. This is the first time that SLIT-ROBO signaling is implicated in human bilateral kidney agenesis., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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., (© 2017 John Wiley & Sons, Ltd.)

Renal agenesis (RA) is one of the more extreme examples of congenital anomalies of the kidney and urinary tract (CAKUT). Bilateral renal agenesis is almost invariably fatal at birth, and unilateral renal agenesis can lead to future health issues including end-stage renal disease. Genetic investigations have identified several gene variants that cause RA, including EYA1 , LHX1 , and WT1 However, whereas compound null mutations of genes encoding α and γ retinoic acid receptors (RARs) cause RA in mice, to date there have been no reports of variants in RAR genes causing RA in humans. In this study, we carried out whole exome sequence analysis of two families showing inheritance of an RA phenotype, and in both identified a single candidate gene, GREB1L Analysis of a zebrafish greb1l loss-of-function mutant revealed defects in the pronephric kidney just prior to death, and F0 CRISPR/Cas9 mutagenesis of Greb1l in the mouse revealed kidney agenesis phenotypes, implicating Greb1l in this disorder. GREB1L resides in a chromatin complex with RAR members, and our data implicate GREB1L as a coactivator for RARs. This study is the first to associate a component of the RAR pathway with renal agenesis in humans., (Copyright © 2017 by the Genetics Society of America.)

Stickler syndrome, a clinically as well as molecularly heterogeneous connective tissue disorder, is predominantly inherited in an autosomal dominant manner and is considered complete penetrant. Previously, mosaicism in Stickler syndrome has been reported in only a few cases. We describe a child with Stickler syndrome due to a novel splice site mutation in COL11A1. Initially, Sanger sequencing of both parents showed normal test results for the mutation. Due to mild phenotypic traits, the father was tested again using a more sensitive method (NGS), and was found to have low-grade mosaicism in various tissue samples (range 7-22% of the DNA). Therefore, we recommend using sensitive genetic testing when mosaicism is suspected. Furthermore, we support previous suggestions of parental testing even when the parents of an affected patient do not have obvious phenotypic signs of Stickler syndrome., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

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.

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.

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5-116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop-gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late "catch-up" growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2)., (© 2015 WILEY PERIODICALS, INC.)

Introduction: Marfan syndrome is an autosomal, dominantly inherited disorder of the connective tissue. We report the clinical data and results of a genetic analysis of a large Danish Marfan family., Methods: Sanger sequencing of FBN1 was initially performed on genomic DNA from the index patient. Subsequently, four affected family members and three non-affected family members were tested for the variant identified in the index patient., Results: A novel variant (c.701G>T) in the FBN1 segregated with Marfan features in the family., Conclusion: In the majority of the family members, this novel variant seems to cause a uniform and very detrimental set of disease characteristics including fatal aortic dissection., Funding: not relevant., Trial Registration: not relevant.

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.

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., (© 2014 Wiley Periodicals, Inc.)

Mosaic PIK3CA-mutations have been described in an increasing number of overgrowth syndromes. We describe a patient with a previously unreported segmental overgrowth syndrome with the mutation, PIKCA3 c.3140A>G (p.His1047Arg) in affected tissue diagnosed by exome sequencing. This PIK3CA-associated segmental overgrowth syndrome overlaps with CLOVES syndrome and fibroadipose hyperplasia but is distinct from each of these entities., (© 2014 Wiley Periodicals, Inc.)

Background: Measurement of milk cobalamin is hampered by the high content of the cobalamin-binding protein haptocorrin, and limited data are available relating trustworthy measures of milk cobalamin to cobalamin status in healthy mothers and their children., Objectives: The objectives were to explore the concentration of cobalamin and haptocorrin in foremilk and hindmilk during the first 9 mo of lactation and to relate these results to biomarkers of an impaired cobalamin status of mother and child., Design: Milk samples from 25 mothers were collected at 2 wk, 4 mo, and 9 mo postpartum for the measurement of cobalamin and haptocorrin. Plasma samples from a larger cohort of lactating mothers (n = 107) and their infants (n = 108) were collected at the same time points for the measurement of cobalamin, holotranscobalamin, total transcobalamin, total haptocorrin, and methylmalonic acid., Results: Median (range) concentrations of cobalamin in hindmilk were 760 (210-1880), 290 (140-690), and 440 (160-1940) pmol/L at 2 wk, 4 mo, and 9 mo, respectively; the respective haptocorrin concentrations were 25 (9-102), 22 (4-100), and 180 (30-460) nmol/L. We found slightly lower values in foremilk. A decrease in milk cobalamin at 4 mo was associated with decreases in plasma cobalamin (P , 0.0001) and holotranscobalamin (P , 0.0001) in the infants. Strong positive associations in paired maternal-infant cobalamin concentrations were found at all time points., Conclusions: Foremilk and hindmilk contained comparable amounts of cobalamin and haptocorrin, but marked changes were observed during 9 mo of lactation. At 4 mo, low concentrations of milk cobalamin mirrored biochemical changes in infants, which suggests an impaired cobalamin status and indicates that nutrition from only mother's milk may not be sufficient for the supply of cobalamin from this age. This trial was registered by the Danish Data Protection Agency at www.datatilsynet.dk/english as 2008-41-2185.

Chronic vitamin B12 (cobalamin) deficiency in the mammalian central nervous system causes degenerative damage, especially in the spinal cord. Previous studies have shown that cobalamin status alters spinal cord expression of epidermal growth factor (EGF) and its receptor in rats. Employing a mouse model of cobalamin-depletion and loading, we have explored the influence of Cbl status on spinal cord expression of cobalamin related proteins, as well as all four known EGF receptors and their activating ligands. Following four weeks of osmotic minipump infusion (n=7 in each group) with cobinamide (4.25nmol/h), saline or cobalamin (1.75nmol/h) the spinal cords were analyzed for cobalamin and for the mRNA levels of cobalamin related proteins and members of the EGF system using quantitative reverse transcription PCR. The median spinal cord cobalamin content was 17, 32, and 52pmol/gr of tissues in cobinamide, saline, and cobalamin treated animals, respectively. Both cobinamide and cobalamin induced a significant decrease in the expression of the lysosomal membrane cobalamin transporter. All four EGF receptors and their activating ligands, except for EGF, were expressed in the spinal cord. Notably, the expression of one of the EGF receptors, HER3, and the ligands heparin-binding EGF-like growth factor, transforming growth factor-α, and neuregulins 1α was increased in cobalamin treated mice. Our studies show that four weeks treatment of mice with cobinamide induces spinal cord cobalamin depletion and that cobalamin loading induces an altered expression pattern of the EGF system thus confirming a spinal cord cross talk between Cbl and the EGF system., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Several studies suggest that the vitamin B12 (B12) transport system can be used for the cellular delivery of B12-conjugated drugs, also in long-term treatment Whether this strategy will affect the endogenous metabolism of B12 is not known. To study the effect of treatment with excess B12 or an inert derivative, we established a mouse model using implanted osmotic minipumps to deliver saline, cobinamide (Cbi) (4.25 nmol/h), or B12 (1.75 nmol/h) for 27 days (n = 7 in each group). B12 content and markers of B12 metabolism were analysed in plasma, urine, kidney, liver, and salivary glands. Both Cbi and B12 treatment saturated the transcobalamin protein in mouse plasma. Cbi decreased the content of B12 in tissues to 33-50% of the level in control animals but did not influence any of the markers examined. B12 treatment increased the tissue B12 level up to 350%. In addition, the transcript levels for methylenetetrahydrofolate reductase in kidneys and for transcobalamin and transcobalamin receptor in the salivary glands were reduced. Our study confirms the feasibility of delivering drugs through the B12 transport system but emphasises that B12 status should be monitored because there is a risk of decreasing the transport of endogenous B12. This risk may lead to B12 deficiency during prolonged treatment.

In our previous studies, one-third of lactating Guatemalan women, infants, and children had deficient or marginal serum vitamin B-12 concentrations. Relationships among maternal and infant status and breast milk vitamin B-12, however, have not, to our knowledge, been investigated in such populations. Our purpose was to measure breast milk vitamin B-12 in Guatemalan women with a range of serum vitamin B-12 concentrations and explore associations between milk vitamin B-12 concentrations and maternal and infant vitamin B-12 intake and status. Participants were 183 mother-infant pairs breastfeeding at 12 mo postpartum. Exclusion criteria included mother <17 y, infant <11.5 or >12.5 mo, multiple birth, reported health problems in mother or infant, and mother pregnant >3 mo. Data collected on mothers and infants included anthropometry, serum and breast milk vitamin B-12, and dietary vitamin B-12. Serum vitamin B-12 concentrations indicated deficiency (<150 pmol/L) in 35% of mothers and 27% of infants and marginal status (150-220 pmol/L) in 35% of mothers and 17% of infants. In a multiple regression analysis, breast milk vitamin B-12 concentration was associated (P < 0.05) with both maternal vitamin B-12 intake (r = 0.26) and maternal serum vitamin B-12 (r = 0.30). Controlling for the number of breastfeeds per day and vitamin B-12 intake from complementary foods, infant serum vitamin B-12 was associated with maternal serum vitamin B-12 (r = 0.31; P < 0.001) but not breast milk vitamin B-12, implicating a long-term effect of pregnancy status on infant vitamin B-12 status at 12 mo postpartum.

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.

Background: Currently, it is unknown whether the decline in plasma cobalamin observed during pregnancy is caused by malabsorption of the vitamin. This study examined cobalamin absorption and markers of cobalamin status during normal pregnancy., Methods: Twenty-seven pregnant Danish women were examined at gestation weeks 13, 24 and 36. The absorption test CobaSorb was performed in all women implying measurement of holotranscobalamin or cyanocobalamin bound to transcobalamin before and after 2 days intake of 3 × 9 μg cobalamin. Serum cobalamin and the two cobalamin binding proteins transcobalamin and haptocorrin, including haptocorrin saturated with cobalamin or analogues, were measured, and so was plasma methylmalonic acid and homocysteine., Results: No change in the uptake of cobalamin was observed throughout pregnancy. Serum cobalamin displayed a gradual decline during pregnancy (p<0.0001), while holotranscobalamin remained unchanged, despite an increase in total transcobalamin (p<0.0001). In accord with these results, total haptocorrin showed a decline from the 1st to 3rd trimester (p=0.007) and cobalamin bound to haptocorrin declined (p<0.0001). Interestingly, the amount of cobalamin analogues attached to haptocorrin remained unchanged. Methylmalonic acid (p=0.002) and homocysteine (p<0.0001) increased during pregnancy., Conclusions: Cobalamin absorption remains unchanged during normal pregnancy, as judged by the CobaSorb test. No change was observed in the biological active holotranscobalamin during pregnancy. Thus, the pregnancy-related decline in cobalamin is caused by alternations in haptocorrin-bound cobalamin. Surprisingly, no pregnancy-related change was observed in the amount of analogues attached to haptocorrin.

Background: Low vitamin B₁₂ concentrations have been associated with higher risks of cognitive impairment, but whether these associations are causal is uncertain. The associations of cognitive impairment with combinations of vitamin B₁₂, holotranscobalamin, methylmalonic acid, and total homocysteine, and with the vitamin B₁₂ transport proteins transcobalamin and haptocorrin, have not been previously studied., Methods: We performed a population-based cross-sectional study of 839 people 75 years old or older. We examined the association of cognitive function as measured by mini-mental state examination scores, with markers of vitamin B₁₂ status. Spearman correlations as well as multivariate-adjusted odds ratios and 95% CIs for cognitive impairment were calculated for extreme thirds of serum concentrations of vitamin B₁₂, holotranscobalamin, methylmalonic acid, total homocysteine, combination of these markers in a wellness score, heaptocorrin, and transcobalamin for all data and with B₁₂ analogs in a nested case-control study., Results: Cognitive impairment was significantly associated with low vitamin B₁₂ [odds ratio 2.3 (95% CI 1.2-4.5)]; low holotranscobalamin [4.1 (2.0-8.7)], high methylmalonic acid [3.5 (1.8-7.1)], high homocysteine [4.8 (2.3-10.0)] and low wellness score [5.1 (2.61-10.46)]. After correction for relevant covariates, cognitive impairment remained significantly associated with high homocysteine [4.85 (2.24-10.53)] and with a low wellness score [5.60 (2.61-12.01)] but not with transcobalamin, haptocorrin, or analogs on haptocorrin., Conclusions: Cognitive impairment was associated with the combined effects of the 4 biomarkers of vitamin B₁₂ deficiency when included in a wellness score but was not associated with binding proteins or analogs on haptocorrin.

Background: Haptocorrin is a vitamin B12-binding protein present in high amounts in different body fluids including human milk. Haptocorrin has previously been shown to inhibit the growth of specific E. coli strains, and the aim of the present study was to elucidate whether the antibacterial properties of this protein may exert a general defense against pathogens and/or affect the composition of the developing microbiota in the gastrointestinal tracts of breastfed infants., Findings: The present work was the first systematic study of the effect of haptocorrin on bacterial growth, and included 34 commensal and pathogenic bacteria to which infants are likely to be exposed. Well-diffusion assays addressing antibacterial effects were performed with human milk, haptocorrin-free human milk, porcine holo-haptocorrin (saturated with B-12) and human apo-haptocorrin (unsaturated). Human milk inhibited the growth of S. thermophilus and the pathogenic strains L. monocytogenes LO28, L. monocytogenes 4446 and L. monocytogenes 7291, but the inhibition could not be ascribed to haptocorrin. Human apo-haptocorrin inhibited the growth of only a single bacterial strain (Bifidobacterium breve), while porcine holo-haptocorrin did not show any inhibitory effect., Conclusions: Our results suggest that haptocorrin does not have a general antibacterial activity, and thereby contradict the existing hypothesis implicating such an effect. The study contributes to the knowledge on the potential impact of breastfeeding on the establishment of a healthy microbiota in infants.

In humans, the cobalamin (Cbl) -binding protein transcobalamin (TC) transports Cbl from the intestine and into all the cells of the body, whereas the glycoprotein haptocorrin (HC), which is present in both blood and exocrine secretions, is able to bind also corrinoids other than Cbl. The aim of this study is to explore the expression of the Cbl-binding protein HC as well as TC in mice. BLAST analysis showed no homologous gene coding for HC in mice. Submaxillary glands and serum displayed one protein capable of binding Cbl. This Cbl-binding protein was purified from 300 submaxillary glands by affinity chromatography. Subsequent sequencing identified the protein as TC. Further characterization in terms of glycosylation status and binding specificity to the Cbl-analogue cobinamide revealed that mouse TC does not bind Concanavalin A sepharose (like human TC), but is capable of binding cobinamide (like human HC). Antibodies raised against mouse TC identified the protein in secretory cells of the submaxillary gland and in the ducts of the mammary gland, i.e. at locations where HC is also found in humans. Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney. By precipitation to insolubilised antibodies against mouse TC, we also showed that >97% of the Cbl-binding capacity and >98% of the Cbl were precipitated in serum. This indicates that TC is the only Cbl-binding protein in the mouse circulation. Our data show that TC but not HC is present in the mouse. Mouse TC is observed in tissues where humans express TC and/or HC. Mouse TC has features in common with both human TC and HC. Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species.