DNA methylation signature classification of rare disorders using publicly available methylation data.

Disease-specific DNA methylation patterns (DNAm signatures) have been established for an increasing number of genetic disorders and represent a valuable tool for classification of genetic variants of uncertain significance (VUS). Sample size and batch effects are critical issues for establishing DNAm signatures, but their impact on the sensitivity and specificity of an already established DNAm signature has not previously been tested. Here, we assessed whether publicly available DNAm data can be employed to generate a binary machine learning classifier for VUS classification, and used variants in KMT2D, the gene associated with Kabuki syndrome, together with an existing DNAm signature as proof-of-concept. Using publicly available methylation data for training, a classifier for KMT2D variants was generated, and individuals with molecularly confirmed Kabuki syndrome and unaffected individuals could be correctly classified. The present study documents the clinical utility of a robust DNAm signature even for few affected individuals, and most importantly, underlines the importance of data sharing for improved diagnosis of rare genetic disorders.

Risks and pregnancy outcome after fetal reduction in dichorionic twin pregnancies: a Danish national retrospective cohort study.

BACKGROUND: Twin pregnancies carry a higher risk of congenital and structural malformations, and pregnancy complications including miscarriage, stillbirth, and intrauterine fetal death, compared with singleton pregnancies. Carrying a fetus with severe malformations or abnormal karyotype places the remaining healthy fetus at an even higher risk of adverse outcome and pregnancy complications. Maternal medical conditions or complicated obstetrical history could, in combination with twin pregnancy, cause increased risks for both the woman and the fetuses. To our knowledge, no previous studies have evaluated and compared the outcomes of all dichorionic twin pregnancies and compared the results of reduced twins with those of nonreduced and primary singletons in a national cohort. These data are important for clinicians when counseling couples about fetal reduction and its implications. OBJECTIVE: This study aimed to describe and compare the risks of adverse pregnancy outcomes, including the risk of pregnancy loss, in a national cohort of all dichorionic twins-reduced, nonreduced, and primary singletons. In addition, we examined the implications of gestational age at fetal reduction on gestational age at delivery. STUDY DESIGN: This was a retrospective cohort study of all Danish dichorionic twin pregnancies, including pregnancies undergoing fetal reduction and a large proportion of randomly selected primary singleton pregnancies with due dates between January 2008 and December 2018. The primary outcome measures were adverse pregnancy outcomes (defined as miscarriage before 24 weeks, stillbirth from 24 weeks, or single intrauterine fetal death in nonreduced twin pregnancies), preterm delivery, and obstetrical pregnancy complications. Outcomes after fetal reduction were compared with those of nonreduced dichorionic twins and primary singletons. RESULTS: In total, 9735 dichorionic twin pregnancies were included, of which 172 (1.8%) were reduced. In addition, 16,465 primary singletons were included. Fetal reductions were performed between 11 and 23 weeks by transabdominal needle-guided injection of potassium chloride, and outcome data were complete for all cases. Adverse pregnancy outcome was observed in 4.1% (95% confidence interval, 1.7%-8.2%) of reduced twin pregnancies, and 2.4% (95% confidence interval, 0.7%-6.1%) were delivered before 28 weeks, and 4.2% (95% confidence interval, 1.7%-8.5%) before 32 weeks. However, when fetal reduction was performed before 14 weeks, adverse pregnancy outcomes occurred in only 1.4% (95% confidence interval, 0.0%-7.4%), and delivery before 28 and 32 weeks diminished to 0% (95% confidence interval, 0.0%-5.0%) and 2.8% (95% confidence interval, 0.3%-9.7%), respectively. In contrast, 3.0% (95% confidence interval, 2.7%-3.4%) of nonreduced dichorionic twins had an adverse pregnancy outcome, and 1.9% (95% confidence interval, 1.7%-2.1%) were delivered before 28 weeks, and 7.3% (95% confidence interval, 6.9%-7.7%) before 32 weeks. Adverse pregnancy outcomes occurred in 0.9% (95% confidence interval, 0.7%-1.0%) of primary singletons, and 0.2% (95% confidence interval, 0.1%-0.3%) were delivered before 28 weeks, and 0.7% (95% confidence interval, 0.6%-0.9%) before 32 weeks. For reduced twins, after taking account of maternal factors and medical history, it was demonstrated that the later the fetal reduction was performed, the earlier the delivery occurred (P<.01). The overall risk of pregnancy complications was significantly lower among reduced twin pregnancies than among nonreduced dichorionic twin pregnancies (P=.02). CONCLUSION: In a national 11-year cohort including all dichorionic twin pregnancies, transabdominal fetal reduction by needle guide for fetal or maternal indication was shown to be safe, with good outcomes for the remaining co-twin. Results were best when the procedure was performed before 14 weeks.

Cell-Based NIPT Detects 47,XXY Genotype in a Twin Pregnancy.

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.

5q11.2 deletion syndrome revisited-Further narrowing of the smallest region of overlap for the main clinical characteristics of the syndrome.

Microdeletions at 5q11.2 are rare. Subjects show a phenotypic spectrum that overlaps CHARGE syndrome and 22q11.2 deletion syndrome. A growing number of subjects present with learning difficulty and/or intellectual disability, immune deficiency, congenital heart malformation, and dysmorphism. DHX29 and IL6ST have been proposed as candidate genes for the development of the major clinical manifestations. We present a new case and narrow down the shortest region of overlap to evaluate possible candidate genes. Our case does not present developmental delay or immune deficiency indicating a reduced penetrance for some of the main clinical manifestations. The shortest region of overlap between subjects with deletions at 5q11.2 is approximately 450 kb (position 54.3-54.7 Mb). The narrowed region comprises 10 protein coding genes, including DHX29. DHX29 is a strong candidate gene for the main features of 5q11.2-microdeletion syndrome; however, our findings suggest a joined impact of several genes as the cause of the syndrome.

Deletion of ERF and CIC causes abnormal skull morphology and global developmental delay.

The ETS2 repressor factor (ERF) is a transcription factor in the RAS-MEK-ERK signal transduction cascade that regulates cell proliferation and differentiation, and pathogenic sequence variants in the ERF gene cause variable craniosynostosis inherited in an autosomal dominant pattern. The reported ERF variants are largely loss-of-function, implying haploinsufficiency as a primary disease mechanism; however, ERF gene deletions have not been reported previously. Here we describe three probands with macrocephaly, craniofacial dysmorphology, and global developmental delay. Clinical genetic testing for fragile X and other relevant sequencing panels were negative; however, chromosomal microarray identified heterozygous deletions (63.7-583.2 kb) on Chromosome 19q13.2 in each proband that together included five genes associated with Mendelian diseases (ATP1A3, ERF, CIC, MEGF8, and LIPE). Parental testing indicated that the aberrations were apparently de novo in two of the probands and were inherited in the one proband with the smallest deletion. Deletion of ERF is consistent with the reported loss-of-function ERF variants, prompting clinical copy-number-variant classifications of likely pathogenic. Moreover, the recent characterization of heterozygous loss-of-function CIC sequence variants as a cause of intellectual disability and neurodevelopmental disorders inherited in an autosomal dominant pattern is also consistent with the developmental delays and intellectual disabilities identified among the two probands with CIC deletions. Taken together, this case series adds to the previously reported patients with ERF and/or CIC sequence variants and supports haploinsufficiency of both genes as a mechanism for a variable syndromic cranial phenotype with developmental delays and intellectual disability inherited in an autosomal dominant pattern.

BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1-/--defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.

A Missense Mutation in RAB28 in a Family with Cone-Rod Dystrophy and Postaxial Polydactyly Prevents Localization of RAB28 to the Primary Cilium.

Purpose: Cone-rod dystrophy (CRD) is a rare hereditary eye disorder that causes progressive degeneration of cone and rod photoreceptors. More than 30 genes, including RAB28, have been associated with CRD; however, only a few RAB28 variants have been reported to be associated with CRD. In this study, we describe two brothers with CRD and a homozygous missense variant, c.55G>A (p.Gly19Arg), in RAB28. Methods: The missense variant was identified as part of a study investigating underlying genetic defects in a large patient cohort (n = 667) using targeted next-generation sequencing of 125 genes associated with retinal dystrophy. Cellular localization of RAB28 and ciliogenesis in patient fibroblasts were investigated by immunofluorescence microscopy. The effect of the missense variant on RAB28 expression level was investigated by quantitative real-time PCR. Results: Two brothers of a consanguineous couple presented with CRD, postaxial polydactyly (PAP), and myopia. Both brothers had a homozygous missense RAB28 variant located in the G1 box of the guanosine triphosphate/guanosine diphosphate binding domain of RAB28. This missense variant caused a considerable reduction of RAB28 localized to the cilia, whereas ciliogenesis seemed unaffected. Conclusions: The missense variant in RAB28 is classified as likely pathogenic with functional effect on protein localization. The combination of retinal dystrophy and PAP are well known from ciliopathies; however, more data are needed to finally conclude that the RAB28 variant described here is the cause of PAP in these brothers.

Generation and characterization of three isogenic induced pluripotent stem cell lines from a patient with Bardet-Biedl syndrome and homozygous for the BBS5 variant.

Bardet-Biedl syndrome (BBS), an autosomal recessive disease, is associated with non-functional primary cilia. BBS5 is part of the protein complex termed the BBSome. The BBSome associates with intra flagellar transport (IFT) particles and mediates trafficking of membrane proteins in the cilium, a process important for cilia-mediated signal transduction. Here we describe the generation of three induced pluripotent stem cell (iPSC) lines, KCi003-A, KCi003-B and KCi003-C from a patient with BBS and homozygous for the disease causing variant c.214G>A, p.(Gly72Ser) in BBS5. The iPSC lines can be used for investigation of IFT in different cell types differentiated from the iPSC line.

[Balanced translocation in a patient with abortus habitualis and normal karyotype].

This is a case report of a 36-year-old woman with un-explained recurrent pregnancy loss, and during her recent pregnancy there were signs of hydrops foetalis. Chorionic villus sampling with array-comparative genomic hybrid-isation revealed an 8.6 Mb duplication of 6q26q27 and a deletion of 14q32.31q32.33. Subsequent genetic exam-inations of the parents with fluorescence in situ hybrid-isation showed a submicroscopically balanced translocation on the patient's chromosomes 6 and 14, which explained her recurrent abortions, and which could not be detected with the conventional genetic testing G-band-karyotyping.

Is MED13L-related intellectual disability a recognizable syndrome?

INTRODUCTION: MED13L-related intellectual disability is characterized by moderate intellectual disability (ID), speech impairment, and dysmorphic facial features. We present 8 patients with MED13L-related intellectual disability and review the literature for phenotypical and genetic aspects of previously described patients. MATERIALS AND METHODS: In the search for genetic aberrations in individuals with ID, two of the patients were identified by chromosomal microarray analysis, and five by exome sequencing. One of the individuals, suspected of MED13L-related intellectual disability, based on clinical features, was identified by Sanger sequencing. RESULTS: All 8 individuals had de novo MED13L aberrations, including two intragenic microdeletions, two frameshift, three nonsense variants, and one missense variant. Phenotypically, they all had intellectual disability, speech and motor delay, and features of the mouth (open mouth appearance, macroglossia, and/or macrostomia). Two individuals were diagnosed with autism, and one had autistic features. One had complex congenital heart defect, and one had persistent foramen ovale. The literature was reviewed with respect to clinical and dysmorphic features, and genetic aberrations. CONCLUSIONS: Even if most clinical features of MED13L-related intellectual disability are rather non-specific, the syndrome may be suspected in some individuals based on the association of developmental delay, speech impairment, bulbous nasal tip, and macroglossia, macrostomia, or open mouth appearance.

Heterozygous loss-of-function variants of MEIS2 cause a triad of palatal defects, congenital heart defects, and intellectual disability.

Deletions on chromosome 15q14 are a known chromosomal cause of cleft palate, typically co-occurring with intellectual disability, facial dysmorphism, and congenital heart defects. The identification of patients with loss-of-function variants in MEIS2, a gene within this deletion, suggests that these features are attributed to haploinsufficiency of MEIS2. To further delineate the phenotypic spectrum of the MEIS2-related syndrome, we collected 23 previously unreported patients with either a de novo sequence variant in MEIS2 (9 patients), or a 15q14 microdeletion affecting MEIS2 (14 patients). All but one de novo MEIS2 variant were identified by whole-exome sequencing. One variant was found by targeted sequencing of MEIS2 in a girl with a clinical suspicion of this syndrome. In addition to the triad of palatal defects, heart defects, and developmental delay, heterozygous loss of MEIS2 results in recurrent facial features, including thin and arched eyebrows, short alae nasi, and thin vermillion. Genotype-phenotype comparison between patients with 15q14 deletions and patients with sequence variants or intragenic deletions within MEIS2, showed a higher prevalence of moderate-to-severe intellectual disability in the former group, advocating for an independent locus for psychomotor development neighboring MEIS2.

Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

Generation of induced pluripotent stem cells, KCi002-A derived from a patient with Bardet-Biedl syndrome homozygous for the BBS10 variant c.271insT.

Bardet-Biedl syndrome (BBS) is genetically heterogeneous with at least 21 genes involved, and BBS10 encodes, together with BBS6 and BBS12, chaperonin-like proteins which are important for the assembly of the multiprotein complex, the BBSome encoded by other BBS genes. Here we describe the successful generation of an induced pluripotent stem cell (iPSC) line KCi002-A from a male with BBS, homozygous for the disease causing variant c.271insT, p.(Cys91fsX95) in BBS10. Resource table.

Generation of induced pluripotent stem cells, KCi001-A derived from a Bardet-Biedl syndrome patient compound heterozygous for the BBS1 variants c.1169T>G/c.1135G>C.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy with a wide range of symptoms including obesity, retinal dystrophy, polycystic kidney disease, polydactyly, hypogonadism and learning difficulties. Here we describe the successful generation of an induced pluripotent stem cell (iPSC) KCi001-A from a BBS patient compound heterozygous for two disease causing BBS1 variants c.1169T>G, p. (Met390Arg)/c.1135G>C, p.(Gly370Arg). Resource table.

[The first Danish patient with a recognisable genetic KBG syndrome].

KBG syndrome is a rare condition characterised by macrodontia of the upper central incisors, distinctive craniofacial findings, short stature, skeletal anomalies, and neurologic involvement including global developmental delay, seizures, and intellectual disability. This is a case report of a seven-year-old boy, who presented with symptoms fulfilling the diagnostic criteria of KBG syndrome, molecularly confirmed by detection of a heterozygous mutation in ANKRD11. To our knowledge, this is the first patient diagnosed with KBG syndrome in Denmark. The aim of this study is to raise awareness of this recognisable syndrome.

Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care.

PURPOSE: Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS. METHODS: In a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations. RESULTS: All anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations. CONCLUSION: Knowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.

Translation and Adaptation of the Genetic Counselling Outcome Scale (GCOS-24) for Use in Denmark.

Outcome measurement in clinical genetics is challenging. Robust outcome measures are needed to provide evidence to support service development within genetic counseling. The Genetic Counselling Outcome Scale (GCOS-24), a Patient Reported Outcome Measure (PROM), was developed in English and validated with clinical genetics patients in the British NHS. This study reports the translation and adaptation of the GCOS-24 for use in Denmark. GCOS-24 was translated and back translated, supervised by an expert committee. Feedback on the first version was collected from genetic counseling patients in qualitative interviews focusing on instructions for use, response options and specific items considered semantically difficult. After further adjustment the adapted and translated version was administered to a second sample of patients, with responses analyzed using descriptive statistics. Eighteen interviews were conducted, and led to adjustment of item wording. Sixty-one patients completed the final version GCOS-24dk. Internal consistency is good (Cronbach's α =0.79), with an acceptable number of missing responses and no floor or ceiling effect observed. GCOS-24 has been successfully translated and adapted for use in a Danish setting. The study confirms the feasibility of local adaptation of patient reported outcome measures and stresses the importance of adaptation, even across quite similar populations and health care systems.

[Hereditary cerebro-oculo-renal syndromes]. / Arvelige cerebrookulorenale syndrome.

Although many congenital diseases present disturbances of the central nervous system, eyes and renal function, only few of these have a defined genetic basis. The first clinical features of cerebro-oculo-renal diseases usually develop in early childhood and deterioration of kidney function and even end-stage kidney disease may occur in a young age. The syndromes should be considered in patients with retarded growth and development, central nervous system abnormalities, impaired vision or blindness and progressive renal failure.

Bardet-Biedl syndrome in Denmark--report of 13 novel sequence variations in six genes.

Bardet-Biedl syndrome (BBS) is an autosomal recessive disease characterized by retinal dystrophy, polydactyly, obesity, learning disabilities, renal involvement, and male hypogenitalism. BBS is genetically heterogeneous with mutations of 14 genes, accounting for approximately 70% of cases. Triallelic inheritance has been suggested in about 5% of cases. Forty-nine unrelated BBS patients were screened for mutations by DHPLC analysis in BBS1, BBS2, BBS4, BBS6/MKKS, BBS10, and BBS12. The selected genes either account for more than 5% of the mutational load or are commonly reported in triallelic inheritance. Eight patients with only one or no BBS mutation were further investigated by single nucleotide polymorphism (SNP) analysis. In total, mutations were detected in 44 patients. Twenty percent had two mutations in BBS1, 18% in BBS2, 4% in BBS9, 43% in BBS10, and 2% in BBS12. Five patients were heterozygous for a sequence variation in BBS6/MKKS. We found eight patients with three sequence variations in two genes, which could be explained by triallelic inheritance, by the prevalence of heterozygous carriers or the third sequence variations representing rare polymorphisms. All changes found in a second BBS gene were amino acid substitutions. Genotype-phenotype correlations suggest a milder phenotype for BBS1 compared to BBS2 and BBS10, which we ascribe to the hypomorphic p.Met390Arg-mutation.