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The German Consortium Hereditary Breast and Ovarian Cancer (GC-HBOC) consists of 23 academic centers striving to provide high-quality regional care for affected individuals and healthy at-risk family members. According to the standard operating procedures defined by the GC-HBOC, a Familial Breast and Ovarian Cancer Center was implemented at the University Medicine Greifswald over a four-year period from 2018 to 2021, despite the COVID-19 pandemic. Genetic analyses were performed in a total of 658 individuals, including 41 males, which paved the way to local annual risk-adapted breast cancer surveillance for 91 women and prophylactic surgery for 34 women in 2021. Our experience in the North Eastern part of Germany demonstrates that it is possible to establish a high-risk breast and ovarian cancer service even in a sparsely populated region. Major facilitators are the interdisciplinary collaboration of dedicated local experts, the support of the GC-HBOC, fruitful clinical and scientific cooperations and the use of technical improvements. As a blueprint, our project report may help to further expand the network of specialized and knowledge-generating care for HBOC families.
The present study was aimed at associating further genes to selected types of laminopathies applying a functional candidate gene approach. Additionally, genotype/phenotype correlations in defined laminopathies were investigated to extend the clinical spectrum and considering practical aspects of molecular genetic analysis in laminopathies. Primary and secondary laminopathies are rare genetic disorders caused by mutations in genes encoding proteins of the nuclear lamina or proteins interacting with the nuclear lamina. So far at least 14 distinct disease phenotypes of primary laminopathies have been found mostly caused by pleiotropic lamin A/C ( LMNA) mutations. Secondary laminopathies can be caused by mutations in other than lamin genes including emerin (STA), lamin associated protein-2 (LAP2) and ZMPSTE24 (ZMPSTE2).
The LINC (Linker of Nucleo- and Cytoskeleton) complex is an evolutionarily conserved complex of nuclear envelope (NE) proteins that forms a direct connection between the nucleoskeleton and cytoskeleton. Primarily, members of two protein families form the complex: SUN and giant nesprin isoforms that reside in the inner and outer nuclear membrane, respectively, thus forming a “bridge” across the NE. Lamin A/C and emerin are additional LINC complex components. Mutations in the genes encoding the LINC complex components have been associated with at least a dozen diseases, the majority of them muscular diseases. Emery-Dreifuss muscular dystrophy (EDMD), an inherited neuromuscular disorder with variable clinical presentation, is one of these diseases. But only around 46% of all EDMD patients are linked to a disease allele. Except of SUN1 and SUN2 all known LINC complex components had been associated with EDMD. Following a functional candidate gene approach the SUN1 and SUN2 genes were sequenced in a cohort of pseudoanonymized 175 EDMD patients without a known mutation and 70 patients with known causative mutations in other LINC components. Based on these results the pathomechanism causing the phenotype in patients with SUN1 or SUN2 mutations was investigated. Autosomal recessive inheritance was observed in one patient with compound heterozygous SUN1 mutations. Patient myoblasts showed defective protein interactions within the LINC complex, altered mRNA expression levels of some LINC components, an enhanced differentiation rate and defects in myonuclear organization. This provides first insights into a new pathomechanism based on weakening of the LINC complex and resulting in disruption of myonuclear alignment. In six patients with known EMD or LMNA mutations additional heterozygous SUN1 or SUN2 mutations modifying the disease have been identified, causing a significantly more severe course. Thus the modifying effect of SUN mutations found in the present study helps to explain the clinical intra- and interfamilial variability observed in EDMD. Further evidence for the influence of mutations in LINC complex components on the molecular pathology of muscular dystrophies comes from a study on primary fibroblasts of a patient suffering Duchenne muscular dystrophy (DMD) and of a patient showing signs of EDMD and Charcot-Marie-Tooth syndrome (CMT). The DMD patient had apart from a mutation in the DMD gene two variants in genes encoding the LINC components nesprin 1α2 and SUN1. The EDMD/CMT patient carried two variants in nesprin 1α1 and SUN2. Fibroblasts of both patients showed changes in cell adhesion, cell migration, senescence and stress response as well as characteristics typical for laminopathies like changes in nuclear shape and NE composition. Mutations in genes encoding LINC complex proteins are also associated with a number of other diseases. Pleiotropic LMNA mutations have also been linked with progeroid syndromes – genetic diseases that mimic clinical and molecular features of aging including Hutchinson Gilford progeria syndrome (HGPS), mandibuloacral dysplasia (MAD), restrictive dermopathy (RD) and atypical Werner’s syndrome (aWS) as well as a couple of overlapping phenotypes. MAD and RD can also be caused by mutations in ZMPSTE24, a gene encoding the ZMPSTE24 metalloproteinase necessary for the processing of prelamin A to mature lamin A. It is expected that insights into the pathomechanism of this group of diseases might provide clues to normal aging process. Analyzing RD and MAD patients for mutations in the ZMPSTE24 gene, some novel mutations have been identified. Based on this results and a review of the literature regarding ZMPSTE24 mutations could be shown that all mutations involved in RD are null mutations, whereas all patients with MAD are compound heterozygotes carrying one loss-of-function mutation and one missense mutation. This shows a clear genotype-phenotype correlation. A further part of this work is the description of the molecular genetics and functional background of a rare, unclassified progeroid syndrome. The clinical course of the affected patient appeared as an accelerated HGPS finally ending up in a delayed RD with overlapping clinical features of MAD. Mutational analysis revealed a homozygous LMNA mutation caused by a partial uniparental disomy of chromosome 1. Immunohistological analyses of tissue samples taken at the beginning and the end of the disease course showed a decreasing amount of lamin A and increasing amounts of DNA double strand breaks. Functional analysis in transfected human normal fibroblasts showed an impaired ability of the mutant lamin A to recruit 53BP1, a component of the DNA repair pathway, to damaged DNA sites. This case provides the first evidence of human lamin A direct involvement in DNA repair and that increased DNA damage is a major pathophysiological factor in progeroid laminopathies.
Genetic heterogeneity and molecular genetic diagnostics in primary and secondary laminopathies
(2008)
Laminopathies are a group of rare genetic disorders caused by mutations in genes encoding proteins of the nuclear lamina. One can distinguish between primary and secondary laminopathies. Primary laminopathies representing at least fourteen disease phenotypes arise through pleiotropic mutations in LMNA - the gene that codes for the A-type lamins A and C, mutations in LMNB1 encoding lamin B1 and mutations in LMNB2 encoding lamin B2. Secondary laminopathies including disease phenotypes also observed in primary laminopathies are caused by genes encoding proteins related to the nuclear lamina like ZMPSTE24 (FACE1), LAP2, LBR and thus reflecting genetic heterogeneity in laminopathies. The goal of the present investigation was to study pleiotropy and genetic heterogeneity in primary and secondary laminopathies by analysis of genotype/phenotype correlations. Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy with conduction disturbances (CMD1A), familial partial lipodystrophy (FPLD), mandibuloacral dysplasia (MAD), progeroid syndrome, atypical Werner syndrome (aWRN), restrictive dermopathy (RD) and Hallermann-Streiff syndrome (HSS) were included as disease phenotypes to look for their association with LMNA (primary laminopathies) and ZMPSTE24 (secondary laminopathies). Additionally, EDMD patients without STA or LMNA mutations were tested for ZMPSTE24 mutations. A functional candidate gene approach was applied using NARF and SREBF1 in patients suffering secondary laminopathies including FPLD, MAD, HGPS and RD, who were excluded from having LMNA and ZMPSTE24 mutations. Finally, practical consequences of the present study have been considered in genetic counseling and prevention of primary and secondary laminopathies. Screening for mutations in LMNA, ZMPSTE24 (FACE1), NARF and SREBF1 was carried out by PCR using intronic primers flanking each of the exons of the genes tested. The PCR products were tested for changes by heteroduplex analysis and directly sequenced by a cycle-sequencing procedure. Each DNA variation found was checked for its frequency in 386 chromosomes of an ethnically matched control population. For primary laminolathies, 249 unrelated individuals suffering EDMD, CMD1A (DCM), FPLD, MAD, HGPS, aWRN, RD, Hallermann-Streiff syndrome or only partially showing clinical features of the afore mentioned disease phenotypes were tested for LMNA mutations. Eighteen independent LMNA mutations were found in 249 unrelated patients resulting in a general detection rate of 7.2% Summary Dissertation 83 Among the 79 unrelated Caucasian patients and seven families suffering EDMD or EDMD-like disease phenotypes, 14 were found with LMNA mutations, including p.E33G, p.R249Q, p.L263P, p.R377H, p.M348I, p.R249W, p.R453W, p.R527P, p.L530R and p.R644C have been found, resulting in a detection rate of 17.7%. Of the ten different mutations, the three mutations p.L263P, p.M348I and p.L530R are novel. The other seven mutations have been reported before to be pathogenic. There is strong evidence that indicates the pathogenicity of the three novel mutations, p.L263P, p.M348I and p.L530R. Firstly, the mutations exchanged evolutionary highly conserved amino acids as shown by orthologous gene comparisons. Secondly, they were not found in 386 alleles of a reference population. Moreover, the mutations are located in the α- helical rod or globular domains of lamins A and C that might lead to the disruption of their nuclear function causing in skeletal and cardiac muscular malfunction. The LMNA p.M348I mutation was found in a Belgian male patient (G-13730) who also carried a STA c.1A>G, p.0 mutation. The STA mutation leading to a loss of emerin has previously shown to be causative for X-linked recessive EDMD and would explain the lack of emerin and a pathogenic effect found in the affected male by itself. But co-segregation of LMNA p.M348I with cardiac conduction disturbances in female family members showed an additional cardiac effect of this mutation to the pathology. This observation is one of the very rare pieces of evidence for digenic (oligo-allelic) pathogenesis in a neuromuscular disease phenotype of laminopathies. It points to related pathogenic mechanisms in EDMD and CMD1A that are not associated with STA and LMNA but with other so far unknown genes functionally related to the nuclear envelope. The known mutation p.R453W of the LMNA gene represents a mutational hot spot. So, it was not unexpectedly found in four unrelated EDMD patients of this study. Other recurrent mutations p.R249Q and p.R377H were found in two patients each. Variable phenotypic expression of the LMNA p.R644C mutation, ranging from no clinical signs to fully expressed EDMD was observed in an Austrian family in the present study. This mutation has reportedly been associated with strikingly diverse phenotypes in unrelated patients including left ventricular hypertrophy, limb girdle muscle weakness, CMD1A, FPLD or atypical lipodystrophy, neuropathy and atypical progeria. But the mechanism of pathogenesis is unknown. The apparent non-penetrance in relatives raises questions about the clinical significance of this missense mutation. However, the observations Summary Dissertation 84 in the present family and in those previously published provide evidence that the risk to express a laminopathy in close relatives is likely to be low but reasonable. Of the 49 unrelated Caucasian patients suffering CMD1A four mutations, p.E161K, c.- 3_+12del, p.Y259C and p.R377H, were found resulting in a detection rate of 8.2%, which did not significantly differ from the 2.5% found in 197 dilated cardiomyopathy patients of an earlier study. This overall low detection rate reflects the wide genetic and environmental heterogeneity of the pathogenesis in dilated cardiomyopathy. Otherwise, LMNA mutations may cause dilated cardiomyopathy in about 5% of the cases. The wide overlapping phenotypic and genetic similarities between Hallermann-Streiff syndrome (HSS) and HGPS, made HSS a good candidate disease for a primary laminopathy caused by LMNA mutations. But there was no co-segregating disease causing mutation identified. Thus, this study excluded HSS for the first time to be associated to LMNA and adds to the molecular genetic differentiation by excluding HSS from primary laminopathies. Among 32 individuals of 12 families suffering restrictive dermopathy, 22 individuals have been found to carry the ZMPSTE24 mutations c.50delA, c.209_210delAT, c.1085 - 1086insT or c.1385T>G. The mutation c.1085 -1086insT is a recurrent mutation that occurred in the present sample with a frequency of 68% in all RD patients with a ZMPSTE24 mutation. Three mutations, c.50delA, c.209_210delAT and c.1385T>G, are novel mutations. Like the c.1085 -1086insT mutation, c.209_210delAT and c.50delA lead to a frame shift, which putatively results in a non-functional truncated peptide. As an additional indication for a pathogenic effect, the novel mutations c.50delA and c.209_210delAT were not found in 386 alleles of a normal reference population. The first ZMPSTE24 missense mutation c.1385T>G (p.L462R) changing a highly conserved amino acid was found in patient from Guinea suffering from a clinically unequivocal case of restrictive dermopathy. The mutation was heterozygous in the patient but also in the healthy mother. A second pathogenic mutation should be expected. This hypothesis could not be proven, as there was no sufficient test material available from the patient and other family members. Moreover, there was no appropriate African (Guinea) reference population available, which could have been used to estimate the frequency of p.L462R. Thus, it cannot be excluded that p.L462R might be a polymorphism or rare non-pathogenic variant in the ethnic group the patient belongs to. Genetic instability in ZMPSTE24 has interfered with the molecular genetic diagnosis of restrictive dermopathy leading to the inability to distinguish between homozygotes and heterozygotes for the ZMPSTE24 mutation c.1085-1086insT. The reason is a repeated Summary Dissertation 85 thymine (T)9 c.1076-1085 in ZMPSTE24 that can cause a slippage of DNA polymerases. By sequencing cDNAs obtained from homozygous wild-type [(T)9], heterozygous [(T)9/(T)10] and homozygous mutant [(T)10] individuals by using regular Taq polymerase (Fermentas) or high fidelity polymerase (Pfu) for the sequencing reaction the genetic instability was quantified. High error rates up to 23% were found if regular Taq polymerase (Fermentas) was used for sequencing while using high fidelity polymerase (Pfu) resulted in error rates of 6.2 % or lower. As a practical consequence, high fidelity polymerase should always be used to distinguish homozygous mutant [(T)10] individuals from heterozygous [(T)9//(T)10] by sequencing. A high percentage of EDMD patients was tested negative for mutations in STA or LMNA (Bonne et al., 2003). Therefore, other genes are supposed to be involved in the molecular pathology of EDMD. ZMPSTE24 was considered as a promising functional candidate gene in this study, as the gene product - the ZMPSTE24 peptide - takes part in the post-translational modification of lamin A. The negative result of the present study points to a rather unlikely association of EDMD with ZMPSTE24. Additionally, NARF can very likely be excluded by this study from being associated with FPLD, MAD, HGPS and RD, while SREBF1 has obviously no association with FPLD. By the present study, diagnostic tools have been established for molecular genetic diagnosis of several very rare primary and secondary laminopathies, which has a direct practical impact on disease management of laminopathies. Now, the molecular definition of the diseases by association with a specific mutation can be used for genotype/phenotype correlation, predictive diagnosis and prenatal diagnosis.
Tetrasomy 9p is a rare chromosomal syndrome and about 30% of known cases exhibit mosaicism. Approximately 50 of the reported cases with tetrasomy 9p mosaicism show a characteristic facial appearance, growth failure, and developmental delay. However, 3 patients with mosaicism for isochromosome 9p and a normal phenotype have also been reported. We report 2 additional cases of clinically normal young females with tetrasomy 9p mosaicism, one of whom also exhibited X chromosome aneuploidy mosaicism leading to an overall of 6 different cell lines. STR analysis performed on this complex mosaic case indicated that the extra isochromosome was of maternal origin while the X chromosome aneuploidy was of paternal origin, indicating a postzygotic event.
Cerebral cavernous malformations are clusters of aberrant vessels that can lead to severe neurological complications. Pathogenic loss-of-function variants in the CCM1, CCM2, or CCM3 gene are associated with the autosomal dominant form of the disease. While interpretation of variants in protein-coding regions of the genes is relatively straightforward, functional analyses are often required to evaluate the impact of non-coding variants. Because of multiple alternatively spliced transcripts and different transcription start points, interpretation of variants in the 5′ untranslated and upstream regions of CCM1 is particularly challenging. Here, we identified a novel deletion of the non-coding exon 1 of CCM1 in a proband with multiple CCMs which was initially classified as a variant of unknown clinical significance. Using CRISPR/Cas9 genome editing in human iPSCs, we show that the deletion leads to loss of CCM1 protein and deregulation of KLF2, THBS1, NOS3, and HEY2 expression in iPSC-derived endothelial cells. Based on these results, the variant could be reclassified as likely pathogenic. Taken together, variants in regulatory regions need to be considered in genetic CCM analyses. Our study also demonstrates that modeling variants of unknown clinical significance in an iPSC-based system can help to come to a final diagnosis.
Cerebral cavernous malformation (CCM) is a neurovascular disease that can lead to seizures and stroke-like symptoms. The familial form is caused by a heterozygous germline mutation in either the CCM1, CCM2, or CCM3 gene. While the importance of a second-hit mechanism in CCM development is well established, it is still unclear whether it immediately triggers CCM development or whether additional external factors are required. We here used RNA sequencing to study differential gene expression in CCM1 knockout induced pluripotent stem cells (CCM1−/− iPSCs), early mesoderm progenitor cells (eMPCs), and endothelial-like cells (ECs). Notably, CRISPR/Cas9-mediated inactivation of CCM1 led to hardly any gene expression differences in iPSCs and eMPCs. However, after differentiation into ECs, we found the significant deregulation of signaling pathways well known to be involved in CCM pathogenesis. These data suggest that a microenvironment of proangiogenic cytokines and growth factors can trigger the establishment of a characteristic gene expression signature upon CCM1 inactivation. Consequently, CCM1−/− precursor cells may exist that remain silent until entering the endothelial lineage. Collectively, not only downstream consequences of CCM1 ablation but also supporting factors must be addressed in CCM therapy development.
Cerebral cavernous malformations (CCM) are low-flow vascular lesions prone to cause severe hemorrhage-associated neurological complications. Pathogenic germline variants in CCM1, CCM2, or CCM3 can be identified in nearly 100% of CCM patients with a positive family history. In line with the concept that tumor-like mechanisms are involved in CCM formation and growth, we here demonstrate an abnormally increased proliferation rate of CCM3-deficient endothelial cells in co-culture with wild-type cells and in mosaic human iPSC-derived vascular organoids. The observation that NSC59984, an anticancer drug, blocked the abnormal proliferation of mutant endothelial cells further supports this intriguing concept. Fluorescence-activated cell sorting and RNA sequencing revealed that co-culture induces upregulation of proangiogenic chemokine genes in wild-type endothelial cells. Furthermore, genes known to be significantly downregulated in CCM3−/− endothelial cell mono-cultures were upregulated back to normal levels in co-culture with wild-type cells. These results support the hypothesis that wild-type ECs facilitate the formation of a niche that promotes abnormal proliferation of mutant ECs. Thus, targeting the cancer-like features of CCMs is a promising new direction for drug development.
Deletions in the CCM1, CCM2, and CCM3 genes are a common cause of familial cerebral cavernous malformations (CCMs). In current molecular genetic laboratories, targeted next-generation sequencing or multiplex ligation-dependent probe amplification are mostly used to identify copy number variants (CNVs). However, both techniques are limited in their ability to specify the breakpoints of CNVs and identify complex structural variants (SVs). To overcome these constraints, we established a targeted Cas9-mediated nanopore sequencing approach for CNV detection with single nucleotide resolution. Using a MinION device, we achieved complete coverage for the CCM genes and determined the exact size of CNVs in positive controls. Long-read sequencing for a CCM1 and CCM2 CNV revealed that the adjacent ANKIB1 and NACAD genes were also partially or completely deleted. In addition, an interchromosomal insertion and an inversion in CCM2 were reliably re-identified by long-read sequencing. The refinement of CNV breakpoints by long-read sequencing enabled fast and inexpensive PCR-based variant confirmation, which is highly desirable to reduce costs in subsequent family analyses. In conclusion, Cas9-mediated nanopore sequencing is a cost-effective and flexible tool for molecular genetic diagnostics which can be easily adapted to various target regions.
Cerebral cavernous malformations (CCMs) are prevalent slow-flow vascular lesions which harbour the risk to develop intracranial haemorrhages, focal neurological deficits, and epileptic seizures. Autosomal dominantly inherited CCMs were found to be associated with heterozygous inactivating mutations in 3 genes, CCM1(KRIT1), CCM2(MGC4607), and CCM3(PDCD10) in 1999, 2003 and 2005, respectively. Despite the availability of high-throughput sequencing techniques, no further CCM gene has been published since. Here, we report on the identification of an autosomal dominantly inherited frameshift mutation in a gene of thus far unknown function, FAM222B(C17orf63), through exome sequencing of CCM patients mutation-negative for CCM1-3. A yeast 2-hybrid screen revealed interactions of FAM222B with the tubulin cytoskeleton and STAMBP which is known to be associated with microcephaly-capillary malformation syndrome. However, a phenotype similar to existing models was not found, neither in fam222bb/fam222ba double mutant zebrafish generated by transcription activator-like effector nucleases nor in an in vitro sprouting assay using human umbilical vein endothelial cells transfected with siRNA against FAM222B. These observations led to the assumption that aberrant FAM222B is not involved in the formation of CCMs.
Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.