Marianne Lodahl

Mutation update on the CHD7 gene involved in CHARGE syndrome.

CHD7 is a member of the chromodomain helicase DNA‐binding (CHD) protein family that plays a role in transcription regulation by chromatin remodeling. Loss‐of‐function mutations in CHD7 are known to cause CHARGE syndrome, an autosomal‐dominant malformation syndrome in which several organ systems, for example, the central nervous system, eye, ear, nose, and mediastinal organs, are variably involved. In this article, we review all the currently described CHD7 variants, including 183 new pathogenic mutations found by our laboratories. In total, we compiled 528 different pathogenic CHD7 alterations from 508 previously published patients with CHARGE syndrome and 294 unpublished patients analyzed by our laboratories. The mutations are equally distributed along the coding region of CHD7 and most are nonsense or frameshift mutations. Most mutations are unique, but we identified 94 recurrent mutations, predominantly arginine to stop codon mutations. We built a locus‐specific database listing all the variants that is easily accessible at www.CHD7.org. In addition, we summarize the latest data on CHD7 expression studies, animal models, and functional studies, and we discuss the latest clinical insights into CHARGE syndrome. Hum Mutat 33:1149–1160, 2012.

Identification of translocation products but not K-RAS mutations in memory B cells from patients with multiple myeloma

Background Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells. Design and Methods The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells. Results Cells with the phenotype of tumor plasma cells (CD38++CD19−CD45−/+CD56−/+/++) or memory B cells (CD38−/CD19+/CD27+) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes (FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K-RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K-RAS mutations were identified. Conclusions The increased expression of a specific “early” oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack “late” oncogene (K-RAS) mutations but express the “early” oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.

Myeloma cell expression of 10 candidate genes for osteolytic bone disease. Only overexpression of DKK1 correlates with clinical bone involvement at diagnosis

Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by interactions between MM cells and the bone marrow microenvironment and is characterized by increased osteoclastic bone resorption and decreased osteoblastic bone formation. Recently, the role of osteoblast inhibition has come into focus, especially the possible role of overexpression of DKK1, an inhibitor of the Wnt signalling pathway. Further, CKS2, PSME2 and DHFR have also been reported as candidate genes for OBD. We studied the gene expression by quantitative reverse transcription polymerase chain reaction of TNFSF11 (RANKL), TNFSF11A (RANK), TNFRSF11B (OPG), CCL3 (MIP1A), CCL4 (MIP1B), PTHR1 (PTHrp), DKK1, CKS2, PSME2 and DHFR in purified, immunophenotypic FACS‐sorted plasma cells from 171 newly diagnosed MM patients, 20 patients with monoclonal gammopathy of undetermined significance and 12 controls. The gene expressions of the analysed genes were correlated with radiographically assessed OBD. Only overexpression of DKK1 was correlated to the degree of OBD. Myeloma cells did not express TNFSF11A, TNFSF11, or TNFRSF11B, and very rarely expressed CCL3 and PTHR11. CCL4, CKS2, PSME2 and DHFR were variably expressed, but the expression of these genes showed no correlation with OBD. In contrast, loss of PSME2 expression in MM plasma cells was significantly correlated with OBD.

Identification of p.A684V Missense Mutation in the WFS1 Gene as a Frequent Cause of Autosomal Dominant Optic Atrophy and Hearing Impairment

Optic atrophy (OA) and sensorineural hearing loss (SNHL) are key abnormalities in several syndromes, including the recessively inherited Wolfram syndrome, caused by mutations in WFS1. In contrast, the association of autosomal dominant OA and SNHL without other phenotypic abnormalities is rare, and almost exclusively attributed to mutations in the Optic Atrophy‐1 gene (OPA1), most commonly the p.R445H mutation. We present eight probands and their families from the US, Sweden, and UK with OA and SNHL, whom we analyzed for mutations in OPA1 and WFS1. Among these families, we found three heterozygous missense mutations in WFS1 segregating with OA and SNHL: p.A684V (six families), and two novel mutations, p.G780S and p.D797Y, all involving evolutionarily conserved amino acids and absent from 298 control chromosomes. Importantly, none of these families harbored the OPA1 p.R445H mutation. No mitochondrial DNA deletions were detected in muscle from one p.A684V patient analyzed. Finally, wolframin p.A684V mutant ectopically expressed in HEK cells showed reduced protein levels compared to wild‐type wolframin, strongly indicating that the mutation is disease‐causing. Our data support OA and SNHL as a phenotype caused by dominant mutations in WFS1 in these additional eight families. Importantly, our data provide the first evidence that a single, recurrent mutation in WFS1, p.A684V, may be a common cause of ADOA and SNHL, similar to the role played by the p.R445H mutation in OPA1. Our findings suggest that patients who are heterozygous for WFS1 missense mutations should be carefully clinically examined for OA and other manifestations of Wolfram syndrome.

In Multiple Myeloma Clonotypic CD38 − /CD19 + /CD27 + Memory B Cells Recirculate Through Bone Marrow, Peripheral Blood and Lymph Nodes

It is believed that myeloma cells are derived from a germinal center (GC) or post GC B cell. The GC B cell can differentiate into both a memory B cell and a plasma cell (PC). In this study, we investigated the recirculating potential of memory B cells clonally related to the myeloma PC (termed clonotypic). The V(H)DJ(H) immunoglobulin gene rearrangement of the myeloma clone was identified for 10 myeloma patients and allele-specific oligonucleotides (ASO) IgH RT-PCR assays were designed for each patient. Memory B cells (CD38- /CD19+ /CD27+) and their subsets defined by the monoclonal antibodies CD62L, CCR6, CXCR4, CXCR5, CCR7 were flow-sorted as single cells and analyzed by ASO RT-PCR analysis. In addition, aspirated peripheral lymph nodes (PLN) of 7 myeloma patients in complete or partial remission were analyzed for the presence of clonotypic cells. Circulating clonotypic memory B cells were identified in PBMNC of 7/10 patients and both CD62L positive and negative clonotypic memory B cells were identified. Furthermore, comparable frequencies of clonotypic cells were found in the CCR6 +/- and CXCR4 +/- memory B cell subsets, whereas all clonotypic memory and later stage B cells were CXCR5 positive. In accordance with their immunophenotype, clonotypic memory B-cells were identified in peripheral blood, bone marrow and PLNs. Clonotypic memory B-cells were present in the majority of myeloma patients and seem to have the same diverse recirculating/homing capacity as normal memory B cells.

Dysregulation of CD47 and the ligands thrombospondin 1 and 2 in multiple myeloma

CD47 and thrombospondin 1 and 2 (TSP1 and TSP2) expression were analysed by real‐time reverse transcription polymerase chain reaction in fluorescence‐activated cell sorted plasma cells (PCs) from patients at consecutive stages of multiple myeloma (MM) development. 80% of MM patients, but only 39% of patients with monoclonal gammopathy of undetermined significance (MGUS) expressed CD47; median expression level increased 10‐fold with progression from MGUS to MM. Elevated TSP1/TSP2 levels occurred in bone marrow cultures from MM patients compared with healthy donors. CD47 and TSP1/TSP2 may have a potential role in the pathophysiology of MM, probably in the interaction between MM PCs and the microenvironment.

Occurrence of dysregulated oncogenes in primary plasma cells representing consecutive stages of myeloma pathogenesis: indications for different disease entities

Summary. This study investigated the expression pattern in primary plasma cells (PCs) of putative oncogenes suggested to be involved in multiple myeloma (MM) development. cDNA archives were generated by global reverse transcription polymerase chain reaction from CD38++/CD19–/CD56–/++ aberrant PCs of a prospective cohort of 96 subjects, including healthy individuals, patients with monoclonal gammopathies of undetermined significance (MGUS), MM and MM with extramedullary manifestations (ExMM). The cDNA archives were analysed quantitatively for expression of the cyclin D1, fibroblast growth factor receptor 3 (FGFR3), C‐MYC, C‐MAF and cyclin D3 oncogenes. In addition, all patients were screened for IGH–MMSET hybrid transcripts. None of the analysed oncogenes was randomly distributed. C‐MYC and cyclin D3 expression increased at the extramedullary transformation stage. Furthermore, C‐MYC and cyclin D3 expression in CD56+ MM was similar to MGUS, whereas CD56– MM was similar to ExMM. FGFR3/IGH–MMSET was only observed among CD56+ MM patients, whereas an increased frequency of C‐MAF dysregulation was seen among CD56– MM. High cyclin D1 expression levels were identified at similar frequencies at all stages, whereas the frequency of patients with low cyclin D1 levels increased during MM development. These data support the stepwise transformation model accumulating genetic alterations and proliferative capacity during MM initiation and development resulting in different clinical entities.

Characterization of potential CD138 negative myeloma "stem cells".

Syndecan-1 is a heparan sulfate proteoglycan expressed by both normal and multiple myeloma (MM) plasma cells. Matsui et al. [1][1] reported the identification of a potential MM “stem cell” resembling less differentiated post germinal center B cells. These cells lacked expression of CD138 but

Expression of HOXA Genes in Patients with Multiple Myeloma

Multiple Myeloma (MM) is an incurable B-cell malignancy characterized by uncontrolled growth of plasma cells (PCs) in the bone marrow. The pathogenesis of MM is complex and still not fully understood. The HOX genes encode a family of homeodomain containing transcription factors which are crucial for embryonic development and differentiation. The HOX genes are also involved in hematopoiesis and have been shown to be dysregulated in leukemia suggesting a role in leukemogenesis. We hypothesized that expression of the HOX genes might also be of importance in MM. We screened FACS-sorted malignant PCs from a panel of 32 MM patients for the expression of HOXA 2, 3, 4, 7, 9, 10, and 11 genes by RT-PCR assays specific for each gene. We found that 9.4% (3/32) of the MM patients expressed the tested HOX genes in their PCs suggesting that HOXA genes are frequently dysregulated and might have an oncogenic potential in MM.

The phenotypic spectrum of dystonia in Mohr–Tranebjaerg syndrome

Mohr–Tranebjaerg syndrome (MTS) is an X‐linked recessive disorder characterized by deafness and dystonia. However the phenotypic expression of dystonia has not been systematically defined. We report clinical, neurophysiological, and ophthalmological data on 6 subjects from 3 Australian kindreds, including 2 with novel mutations, together with a systematic review of the literature, in order to define the phenotypic expression of dystonia. Profound hearing impairment in affected males develops by infancy and precedes the development of dystonia, which varies in time of onset from the first to the sixth decades, with a peak in the second and third decades. Dystonia in MTS tends to be focal, segmental, or multifocal in distribution at onset, with a predilection for the upper body, variably involving the head, neck, and upper limbs. The majority of patients have progression or generalization of their dystonia regardless of age of onset. Within our 3 kindreds, we observed relative intrafamilial homogeneity but interfamilial variation. The median time to the development of moderate‐severely disabling dystonia in these subjects was 11 years. Associated features included progressive cognitive decline, pyramidal signs, and in 1 patient, gait freezing and postural instability. Optic atrophy and cortical visual impairment were both observed. We report for the first time a female patient who developed multiple disabling neurological complications of MTS. Our findings more clearly define and expand the phenotype of both the dystonia and other neurological features of MTS and have implications for the diagnosis and management of this condition.