Inhibition of osteoclastogenesis by RNA interference targeting RANK

Ruofan Ma
Jie Xu
†
Bin Dong
Max Daniel Kauther
Marcus Jäger
Christian Wedemeyer
Inhibition of osteoclastogenesis by RNA interference targeting RANK
R E S E A R C H A R T I C L E Open Access
Background: Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK) ligand (RANKL) as well as the macrophage colony-stimulating factor (M-CSF). The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption.Methods: Three pairs of short hairpin RNAs (shRNA) targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs) using the optimal shRNA by targeting RANK. Results: Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p < 0.01). shRANK-3 also brought about a marked inhibition of osteoclast formation and bone resorption as demonstrated by tartrate-resistant acid phosphatase (TRAP) staining and osteoclast resorption assay. The results of our study show that retrovirus-mediated shRANK-3 suppresses osteoclast differentiation and osteolysis of BMMs.Conclusions: These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

Background

Osteoclasts promote bone resorption in metabolic, degenerative and neoplastic bone disorders. Clinically, the mechanisms inhibiting osteoclast formation are potentially important for preventing bone resorption in these bone disorders. Recent studies on cellular and molecular interactions leading to osteolysis have demonstrated a critical role for the receptor activator of nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) and its ligand (RANK/RANKL) [1]. RANKL is expressed on the surface of stromal cells and osteoblasts. It binds to RANK which is expressed on osteoclast progenitors to induce and promote further differentiation into mature osteoclasts [2]. Stromal cells/ osteoblasts also secrete osteoprotegerin (OPG), a soluble glycoprotein of the tumor necrosis factor receptor super-family. OPG acts as a decoy receptor for RANKL, competing against RANK and thereby inhibiting osteoclast differentiation [3]. In addition, the macrophage colony-stimulating factor (M-CSF) is also produced by stromal cells and induces RANKL-like effects in osteoclasts. It is essential for the survival and proliferation of macrophages and the regulation of osteoclastogenesis [4]. Furthermore, various cytokines or hormones influence the complex osteoclast differentiation by regulating expression of RANKL, M-CSF and OPG on stromal cells or osteoblasts [5].

However, the function of RANK is not limited to cell differentiation. As a member of the tumor necrosis factor receptor (TNFR) family RANK is expressed on the surface of osteoclast progenitor cells and plays an important role in bone homeostasis. RANK transduces intracellular signals upon ligand binding by recruiting various adaptor proteins through specific motifs in the cytoplasmic domain [5][6][7][8][9][10][11]. Thus, the central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies.

The aim of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages (BMMs) on osteoclast differentiation and bone resorption. We also studied the extent of osteoclast inhibition by targeting RANK with retrovirus-mediated shRNA.

Methods

Isolation and culture of bone marrow macrophages

All experimental procedures involving animals were performed in compliance with the regulations of the Guide for the Care and Use of Laboratory Animals (NIH publications Nos. 80-23, revised 1996). Furthermore, the procedures complied with the institutional ethical guidelines for animal experiments and approval was received from the institutional review board.

The femora of 5-week-old BALB/c mice fresh cadavers were aseptically removed and dissected free of adhering tissues. The bone ends were cut off with scissors and the marrow cavity was flushed slowly by injecting Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA). The bone marrow cells were harvested from one end using a sterile needle. 5 × 10 6 of these cells were adhered to tissue culture dishes in 10 ml of DMEM containing 10% fetal bovine serum (FBS; Gibico, BRL, Germany), 100 IU/ml penicillin G (Gibico) and 100 μg/ ml streptomycin (Gibico) with 100 ng/ml M-CSF (PerproTech, London, UK). Three days later, the floating cells were removed and the attached cells were harvested by treatment with phosphate buffer solution (PBS) and used as bone marrow macrophages (BMMs).

CD14 and CD34 immunohistochemistry of BMMs

The sections adhered with cells were harvested and washed with PBS, fixed with 95% ethanol for 30 minutes at 25°C, and then washed three times with cold PBS. The sections were placed in a pressure cooker for antigen retrieval using citrate buffer pH6 for 10 minutes. They were then incubated at room temperature and washed with distilled water. After washing, the sections were placed in hydrogen peroxidise 3% for 6 minutes to block endogenous peroxide, washed with water three times and finally with Tris-buffered saline (TBS) for 10 minutes to eliminate non-specific staining. The excess TBS was removed from the slides before incubation with primary antibody.

The immunohistochemical procedure and preparation of controls were carried out according to the manufacturing company's standards and guidelines. The controls were used to assess the specificity of the reaction as follows: pre-incubation of the cells with the peptide in order to obtain the first antibody, incubation of the cells with an irrelevant primary antibody of the same isotype followed by the second antibody. The sections were incubated with the primary antibodies (CD14 (Santa Cruz, CA, USA, sc-73794) [1:250] and CD34 (Santa Cruz, sc-7045) [1:250]) for 30 minutes at room temperature, washed with TBS and incubated with link antibody for 15 minutes each. Then the sections were washed with TBS and incubated with labeled Streptavidin-biotin (LSAB) for 15 minutes at room temperature, washed again with TBS and incubated with diaminobenzidine (DAB) and substrate chromogen system for 5 minutes at room temperature which resulted in brown-coloured precipitates at the antigen site ( Figure 1A-B).

Cloning into retroviral vectors, selection of the optimal shRNA and stable infection RANK small hairpin RNA (shRNA) was designed and cloned into the pSUPER-retro-puro retroviral vector (Oligoengine, Seattle, Wash., USA). Three shRNAs were chosen based on the sequence of the mouse RANK gene (GenBank gi: NM_ 009399). They covered different regions of the RANK sequence and showed no homology with non-RANK sequences. A scrambled shRNA was used as a control. The target sequences and corresponding oligonucleotide sequences for the four shRNAs, designated shRANK-1, shRANK-2, shRANK-3 and scramble shRNA, are shown in Table 1. Transfection of cells was carried out with Lipofectamine 2000 TM reagent (Invitrogen). Control cells were treated with pSUPER-retro-puro retroviral vector. The relative RANK mRNA and protein levels were determined by Real-time polymerase chain reaction (RT-PCR) and Western blot analyses respectively. The optimal shRANK or vector plasmid and the packaging plasmid PIK were transfected into 293FT cells using the calcium phosphate precipitation method [12,13]. Competent retroviruses were collected 48 hours after transfection. The retroviruses harboring shRANK were transfected into BMMs. Subsequently, the cells were passaged and harvested after treatment in 0.625 μg/ml puromycin (Sigma, St. Louis , USA) medium for three days. Meanwhile, pSUPER-GFPretro-puro retroviral vector was used to generate shRANK retrovirus. After transfection, BMMs were passaged and purified by puromycin. The effect of gene silence was also investigated by detection of GFP expression with fluorescence microscopy.

Isolation of RNA and real time RT-PCR analysis

Total RNA was isolated from cultured cells and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The yield and purity of the RNA was controlled photometrically. RT-PCR was performed in the ABI PRISM W 7000 Sequence Detection System (Applied Biosystems, Foster, USA) using the SYBR Green I master Mix (Applied Biosystems) according to the manufacturer's instructions. RT-PCR of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), a housekeeping gene, served as a control. Primers used in this study were as follows: GAPDH, 5'-CCAATGTGTCCGTCGTGGAT-3' (forward) and 5'-TGCTGTTGAAGTCGCAGGAG -3' (reverse); RANK, 5' -CTGCCTCTGGGAACGTGACT -3' (forward) and 5'-GCGAGGTCTGGCTGACATAC-3' (reverse). Two pairs of primers were added in the action tube for RT-PCR. The PCR cycle conditions were as follows: 2 minutes at 95°C, 30 seconds at 95°C, 35 seconds at 60°C, 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C. Each PCR procedure included a negative control reaction without a template.

Western blot analysis

The cells were washed three times with ice-cold PBS and lysed in RIPA lysis buffer (Sigma, St. Louis, MO, USA). The lysates with Sodium dodecyl sulphate (SDS) buffer were boiled for 5 minutes and separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). They were then transferred to a 0.45 μm polyvinylidene difluoride membrane (PVDF; Millipore, Bedford, USA) and incubated with RANK antibodies (Santa Cruz, CA, USA) at a dilution of 1/3000 and Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Santa Cruz) at a dilution of 1/6000. The HRP substrate was observed on the PVDF membrane. After three washes, the PVDF membrane was incubated with β-actin antibody (Santa Cruz) and HRP-conjugated antibody. The HRP substrate was observed again. For densitometric analyses, blots were scanned and quantified using Quantity One analysis software (Bio-Rad, Hercules, USA), and the results were expressed as the percentages of β-actin immunoreactivity.

In vitro osteoclastogenesis assays and bone resorption assay

BMMs or retrovirally infected BMMs were cultured in 24-well tissue culture plates (5 × 10 4 cells/well) in DMEM containing 10% FBS in the presence of 30 ng/ml M-CSF and 100 ng/ml RANKL (PeproTech, London, UK). Osteoclastogenesis began on the sixth day and the cultures were stained for tartrate-resistant acid phosphatase (TRAP) on the ninth day using a kit (Sigma, St. Louis, MO, USA) in accordance with the manufacturer`s  Table 1 Target sequences and corresponding oligonucleotide sequences for the four shRNAs, designated shRANK-1, shRANK-2, shRANK-3 and scramble shRNA Only TRAP-positive cells with more than three nuclei were counted optically. The values were expressed as means and standard deviations of triplicate cultures. Bone resorption assays were performed using osteoclasts generated on mice bone slices from infected or uninfected BMMs as described previously. The bone slices were harvested on the ninth day. Osteoclastic resorption was quantified by measuring the area of resorption pits with digital image analysis software (Image-pro plus 6.0, Media Cybernetics, USA). The percentage of resorption was calculated by dividing the resorbed area by the total area of the imaged bone slice surface.

Statistical analysis

Data are expressed as mean ± standard deviation. The statistical significance of differences between the experimental and the control groups was evaluated by independent Student's t test. Differences between the groups of the optimal shRANK selection were analyzed using analysis of variance (ANOVA) with SPSS W 16.0 software (SPSS Inc, Chicago, IL). P <0.05 was considered to be statistically significant.

Results and discussion

As shown in Figure 1, nearly all cultured BMMs expressed the monocyte/macrophage surface markers CD14 (94.3-0.4%) and CD34 (90.6-3.5%). BMMs also expressed CD11b (94.8-2.9%) and F4/80 (93.1-3.0%), which were the special surface markers of monocyte/ macrophage [14]. Flow cytometric analysis on day 7 showed similar patterns of surface marker expression (data not shown).

RANK mRNA inhibition by RT-PCR

The inhibition rates of pshRANK-1, pshRANK-2 and pshRANK-3 were 43.4%, 57.3% and 65.8% (p < 0.01) respectively compared with the control plasmid pSUPERretro-puro ( Figure 2).

RANK protein inhibition by Western blot

The inhibition rates of pshRANK-1, pshRANK-2 and pshRANK-3 were 59.4%, 63.3% and 88.3% (p < 0.01) respectively compared with the control plasmid pSUPERretro-puro ( Figure 3).

Stable silencing of the RANK in BMMs by retrovirusmediated shRNA

Subsequently, the effect of shRANK-3 on infected BMMs was studied to determine the biological influence of RANK on osteoclastogenesis in BMMs. Western blot was used to evaluate inhibition of RANK expression in stable cells. Expression was reduced by about 80.7% (p < 0.01), as compared with uninfected BMM cells ( Figure 4). Meanwhile, the levels of RANK protein were significantly reduced in the 3rd-passage cells, which were used in all experiments in this study. The expression of GFP was continuously monitored with fluorescence microscopy to test whether the cells stably express GFP-retro-puro retrovirus. The 10th-passage cells were detected to stably express GFP protein.

RANK silencing in BMMs abolishes osteoclast differentiation and osteolysis

We performed functional assays to determine the effect of RANK inhibition on osteoclast differentiation and osteolysis. Infected BMM cells or BMM cells were treated with RANKL (100 ng/ml) and M-CSF (30 ng/ml) for an additional nine days and subjected to TRAP staining and osteoclast resorption assay. TRAP + cells with more than three nuclei were counted and the area of osteoclast resorption was measured with a scanning electron microscope (SEM). We noted a reduction in the number of TRAP + multinucleated cells in infected BMMs (6.8 ± 1.64) compared with BMMs (82 ± 4.64) (p < 0.05) ( Figure 5 A-C). Furthermore, a significant reduction in resorption areas on the bone slices ( Figure 5 D-F) in infected BMMs (16.6% ± 2.70%) compared with BMMs (2.8% ± 0.84%) (p < 0.05) was observed, indicating profound inhibition of osteoclastic bone resorption. Meanwhile, the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy ( Figure 5 G, H). The difference is obvious.

Discussion

Osteoclasts (OCs) are multinucleated cells derived from circulating osteoclast precursor cells (OCPs) of the monocyte/macrophage lineage. They represent the only cell type capable of bone resorption [5]. Osteoclasts promote bone resorption in metabolic, degenerative and neoplastic bone disorders. Excess osteoclast activity in osteolysis may involve not only generation and activation of OCs, but also increased recruitment of OCPs. The increased bone resorption in osteolysis is thought to be mediated via numerous pro inflammatory cytokines. These cytokines are considered to act primarily via a common final pathway involving members of the TNF receptor-ligand family: The RANKL and its corresponding RANK receptor that play a crucial role in osteoclast differentiation and activation, and OPG, the physiological inhibitor of RANKL [15]. In turn, excess osteoclast activity is associated with the disorders of the TNF receptor-ligand family that ultimately lead to metabolic, degenerative and neoplastic bone disorders. Clinical treatments are needed that can inhibit excess osteolysis in an inflammatory microenvironment. Given that RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis, we tested the hypotheses that inhibition of RANK expression by RNA silencing would reduce the number of osteoclasts and the activity of bone erosion. The limitation of our study is that BMMs culture experiments have a limited perspective of time as we analyzed the BMM cells for only nine days. As metabolic, degenerative and neoplastic bone disorders are processes which can take years, the reactions by osteoclasts might change in the course of time. The correlational research in vivo should be carried on in future studies. Nevertheless, we demonstrated that the difference in osteoclast number and bone erosion between the study groups was significant.

Our data showing inhibition of RANK expression by successful silencing of RNA, which can inhibit specific gene expression, suggests that we can obtain the optimal shRNA-silencing RANK. The inhibition rates of pshRANK-1, pshRANK-2 and pshRANK-3 by Western Blot were 59.4%, 63.3% and 88.3% (p < 0.01) respectively compared with the control plasmid pSUPER-retro-puro, which is consistent with the result of Real-time PCR. Thus, shRANK-3 was identified as the optimal sequence silencing RANK.

Our study shows that inhibition of RANK expression suppresses osteoclast differentiation, thus leading to reduction of bone resorption. We found that RANK gene-silencing using retrovirus-mediated shRNA can significantly suppress RANK expression of BMMs. The inhibition rates of RANK protein by Western Blot was about 80.7% (p < 0.01). TRAP staining and SEM reveal that the osteoclastogenesis of infected BMMs is significantly reduced, compared with uninfected BMMs.

We are not aware of any study showing a similar effect of retrovirus-mediated gene therapy with pshRANK on osteoclastogenesis in BMMs. Although OPG fusion protein and OPG have been used successfully to prevent osteolysis, OPG may bind TNF related apoptosisinducing ligand in addition to RANKL and thus act as a cancer survival factor [16][17][18]. Clinical trials with a monoclonal antibody against RANKL showed efficacy and anti-resorptive activity [19], but sensitization to monoclonal antibodies`therapeutics poses significant risk to the patient and may blunt the efficacy of these therapies [20]. In addition, antibodies against OPG fusion protein harbor the potential risk of cross-reacting with and neutralizing endogenous OPG.

Numerous studies have shown that the RANK/ RANKL pathway is central to cellular and molecular mechanisms associated with the process of osteolysis [4,15,18].

Subsequently, a gene-silenced RANK gene leads to an imbalance of a variety of downstream signaling pathways which are required for osteoclast development [21]. RANKL binding to RANK induces the trimerization of RANK and TNF receptor-associated factors 6 (TRAF6), which leads to the activation of mitogenactivated kinases (MAPK). TRAF6 adaptor protein binds to the intracellular part of the activated RANK receptor and starts the NF-κB pathway, which finally leads to activation of osteoclastogenesis [22]. Different members of the MAPK family, such as p38-MAPKα and p38-MAPKβ, are known to be involved in osteoclastogenesis [23]. Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis is mediated by receptor activator of NF-κB ligand (RANKL) [23]. Furthermore, an influence on bone metabolism is caused by the induction of the phoshatidylinositol (PI) metabolism, which is mediated by the c-Src kinase. The targeted disruption of the c-Src proto-oncogene leads to osteopetrosis in mice [24]. Following notable advances in the understanding of intracellular signaling pathways of RANK, therapeutic drugs specifically targeting downstream signaling molecules have been developed including Interferon-β, γ, p38 inhibitor (SB203580, FR167653), JNK inhibitor (SB600125), IKappaB kinase (IKK) and NEMO binding domain (NBD peptide) inhibitor, NF-κB inhibitor (NF-κB decoy), Calcineurin inhibitor (Cyclosporin A, FK 506), NFAT inhibitor (VIVIT peptide), PI3K inhibitor (Wortmannin, LY290442) [25][26][27][28][29][30]. The limitation of these approaches lies in the lack of specificity of these compounds, since these intracellular signaling pathways are also important for the function and homeostasis of other cells and tissues. Furthermore, RANK is expressed by other types of cells (inflammatory cells and endothelial cells) and plays a role in other physiological processes such as inflammation and angiogenesis. This requires the development of a sophisticated cell-specific or tissue-specific drug delivery system to prevent or mitigate their adverse effects [31].

RANK suppression in the RANKL/RANK system appears to be a promising target for potential therapies in the treatment of osteolysis. RNA interference to inhibit specific gene expression has been shown to be an effective and promising technology for both basic science research and therapeutic intervention [32,33].

Conclusions

Our results suggest that retrovirus-mediated pshRANK suppresses RANK expression of BMMs which further inhibits osteoclastogenesis of BMM treated with M-CSF and RANKL. These findings may provide novel therapeutic strategies for the management of osteolysis. Safety and ethics are areas of concern for employing this therapeutic intervention in humans. In future studies, we would like to investigate the effects of retrovirusmediated pshRANK on the inhibition of osteoclast differentiation and osteolysis in vivo.

Figure 1
1CD14 (A) and CD34 (B) immunohistochemistry of isolated bone marrow macrophages (BMMs).

5'--3' AGCTTAAAAATGGGCTTCTTCTCAGATGTTCTCTTGAAACATCTGAGAAGAAGCCCA GGG shRANK-3 gcgctgacagctaatttgt Forward: 5'--3' GATCCCCGCGCTGACAGCTAATTTGTTTCAAGAGAACAAATTAGCTGTCAGCGC TTTTTA Reverse: 5'--3' AGCTTAAAAAGCGCTGACAGCTAATTTGTTCTCTTGAAACAAATTAGCTGTCAGCGC GGG scrambled shRNA Forward: 5'--3' GATCCCCGCCAGCTTAGCACTGACTCTTCAAGAGAGAGTCAGTGCTAAGCTGGCTTTTT A Reverse: 5'--3' AGCTTAAAAAGCCAGCTTAGCACTGACTCTCTCTTGAAGAGTCAGTGCTAAGCTGGCGGG instructions. TRAP-positive cells appeared red to purple.

Figure 2
2RANK gene expression inhibited by shRNAs and the inhibition rate of psh RANK-1, psh RANK-2 and psh RANK-3. Lane 1: pSuper-retro-puro; lane 2: pscrambled shRNA; lane 3: psh RANK-1; lane 4: psh RANK-2; lane 5: psh RANK-3.

Figure 3
3Western blot for RANK (A) and the inhibition rate of psh RANK-1, psh RANK-2 and psh RANK-3 (B) in BMMs.Figure 1 RANK gene expression inhibited by shRNAs and the inhibition rate of psh RANK-1, psh RANK-2 and psh RANK-3. Lane 1: Psuper-retro-puro; lane 2: pscrambled shRNA; lane 3: psh RANK-1; lane 4: psh RANK-2; lane 5: psh RANK-3.

Figure 4
4RANK silencing in BMMs by stable transduction by retroviruses. BMMs stably transduced with shRNA against the RANK were analyzed for RANK protein levels by Western blot(A, B); β-actin served as a loading control.

Abbreviations NF-kappa B: Nuclear factor`kappa-light-chain-enhancer`of activated B-cells; RANK: Receptor activator of NF-kappa B; RANKL: Receptor activator of NF-kappa B; RNA: Ribonucleic acid; shRNA: Short hairpin ribonucleic acid; MCSF: Macrophage colony-stimulating factor; BMM: Bone marrow macrophages; TRAP: Tatrate-resistant acid phosphatase; OPG: Osteoprotegerin; TNFR: Tumor-necrosis factor receptor; DMEM: Dulbecco`s modified eagle`s medium; PBS: Phosphate buffered solution; TBS: Tris-buffered saline; DAB: Diaminobenzidine; RT-PCR: Realtime-polymerase chain reaction; GAPDH: Glyceralaldehyde-3-phosphate; SDS: Sodium dodecyl sulphate dehydrogenase; HRP: Horseradish peroxidase; ANOVA: Analysis of variance; OC: Osteoclast; OCP: Osteoclast precursor cell; TRAF 6: TNF receptor-associated factor 6; MAPK: Mitogen-activated protein kinase; PI: Phosphatidyl inositol; JNK: c-Jun N-terminal kinase; IKK: IkappaB kinase; NFAT: Nuclear factor of activated T-cells; NBD: NEMO binding domain; PI3K: Inhibitor phosphoinositide 3-kinases. Competing interests This study was financed by the Research Fund of Social Development and the Science and Technology Research of Guangdong Province of China (2009B030801025, 2011B031800156) and by the University of Duisburg-Essen with money given in addition to the DFG (Deutsche Forschungsgemeinschaft) grant of Christian Wedemeyer and Max Daniel Kauther (WE 3634/1-1 and WE 3634/1-2).

Figure 5
5Inhibition of osteoclastogenesis by RANK silencing. (A, B) Osteoclast differentiation of BMMs as identified by TRAP staining. (C) Quantification of the data. The multinucleated TRAP-positive cells (>3 nuclei) in eight random view areas (at 200× magnification) in each of the five replicates were counted. Osteoclast resorption was quantified using toluidine blue staining (at 400× magnification) (D,E,F). Comparison of the resorption area of bone slices between untreated and treated BMM cells was evaluated by Scanning Electron Microscopy (at 400× magnification) (G, H).
http://www.biomedcentral.com/1471-2474/13/154
AcknowledgementsThe authors thank Mrs. Kaye Schreyer for editorial assistance and proof reading.Authors' contribution RM and JX carried out the molecular genetic studies and made substantial contributions to the concept and the design of the study, the analysis and the interpretation of the data and drafting of the manuscript. BD carried out the scientific assays and participated in the drafting and critical review of the manuscript. MK participated in the design of the study, performed the statistical analysis and made a critical review of the manuscript. CW conceived the study, participated in its design and coordination and contributed to the drafting of the manuscript. All authors read and approved the final manuscript.Submit your next manuscript to BioMed
Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. T Suda, N Takahashi, N Udagawa, E Jimi, M T Gillespie, T J Martin, Endocr Rev. 203Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ: Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev Jun 1999, 20(3):345-357.

Regulation of the differentiation and function of osteoclasts. T J Chambers, J Pathol. 1921Chambers TJ: Regulation of the differentiation and function of osteoclasts. J Pathol 2000, 192(1):4-13.

The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. L C Hofbauer, S Khosla, C R Dunstan, D L Lacey, W J Boyle, B L Riggs, J Bone Miner Res. 151Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL: The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 2000, 15(1):2-12.

Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors. F Arai, T Miyamoto, O Ohneda, T Inada, T Sudo, K Brasel, T Miyata, D M Anderson, T Suda, J Exp Med. 19012Arai F, Miyamoto T, Ohneda O, Inada T, Sudo T, Brasel K, Miyata T, Anderson DM, Suda T: Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors. J Exp Med 1999, 190(12):1741-1754.

Osteoclast differentiation and activation. W J Boyle, W S Simonet, D L Lacey, Nature. 4236937Boyle WJ, Simonet WS, Lacey DL: Osteoclast differentiation and activation. Nature 2003, 423(6937):337-342.

All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. J Y Chung, Y C Park, H Ye, H Wu, J Cell Sci. 115Pt 4Chung JY, Park YC, Ye H, Wu H: All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. J Cell Sci 2002, 115(Pt 4):679-688.

The TNF and TNF receptor superfamilies: integrating mammalian biology. R M Locksley, N Killeen, M J Lenardo, Cell. 1044Locksley RM, Killeen N, Lenardo MJ: The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 2001, 104(4):487-501.

Regulatory roles and molecular signaling of TNF family members in osteoclasts. X Feng, Gene. 3501Feng X: Regulatory roles and molecular signaling of TNF family members in osteoclasts. Gene 2005, 350(1):1-13.

Functional identification of three receptor activator of NF-kappa B cytoplasmic motifs mediating osteoclast differentiation and function. W Liu, D Xu, H Yang, H Xu, Z Shi, X Cao, S Takeshita, J Liu, M Teale, X Feng, J Biol Chem. 27952Liu W, Xu D, Yang H, Xu H, Shi Z, Cao X, Takeshita S, Liu J, Teale M, Feng X: Functional identification of three receptor activator of NF-kappa B cytoplasmic motifs mediating osteoclast differentiation and function. J Biol Chem 2004, 279(52):54759-54769.

RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. N Nakagawa, M Kinosaki, K Yamaguchi, N Shima, H Yasuda, K Yano, T Morinaga, K Higashio, Biochem Biophys Res Commun. 2532Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Yano K, Morinaga T, Higashio K: RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res Commun 1998, 253(2):395-400.

RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-beta. H Takayanagi, S Kim, K Matsuo, H Suzuki, T Suzuki, K Sato, T Yokochi, H Oda, K Nakamura, N Ida, E F Wagner, T Taniguchi, Nature. 4166882Takayanagi H, Kim S, Matsuo K, Suzuki H, Suzuki T, Sato K, Yokochi T, Oda H, Nakamura K, Ida N, Wagner EF, Taniguchi T: RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-beta. Nature 2002, 416(6882):744-749.

Applications of RNA interference in cancer therapeutics as a powerful tool for suppressing gene expression. S He, D Zhang, F Cheng, F Gong, Y Guo, Mol Biol Rep. 36He S, Zhang D, Cheng F, Gong F, Guo Y: Applications of RNA interference in cancer therapeutics as a powerful tool for suppressing gene expression. Mol Biol Rep 2009, 36:2153-2163.

Downregulation of BMI-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells. L Qin, X Zhang, L Zhang, Y Feng, G X Weng, M Z Li, Q L Kong, C N Qian, Y X Zeng, M S Zeng, D F Liao, L B Song, Biochem Biophys Res Commun. 3713Qin L, Zhang X, Zhang L, Feng Y, Weng GX, Li MZ, Kong QL, Qian CN, Zeng YX, Zeng MS, Liao DF, Song LB: Downregulation of BMI-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells. Biochem Biophys Res Commun 2008, 371(3):531-535.

Generation of murine bone marrow derived macrophages in a standardised serum-free cell culture system. K Eske, K Breitbach, J Köhler, P Wongprompitak, I Steinmetz, J Immunol Methods. 3421-2Eske K, Breitbach K, Köhler J, Wongprompitak P, Steinmetz I: Generation of murine bone marrow derived macrophages in a standardised serum-free cell culture system. J Immunol Methods 2009, 342(1-2):13-19.

The biology of aseptic osteolysis. G Holt, C Murnaghan, J Reilly, R M Meek, Clin Orthop Relat Res. 460Holt G, Murnaghan C, Reilly J, Meek RM: The biology of aseptic osteolysis. Clin Orthop Relat Res 2007, 460:240-252.

Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis. J R Canon, M Roudier, R Bryant, S Morony, M Stolina, P J Kostenuik, W C Dougall, Clin Exp Metastasis. 252Canon JR, Roudier M, Bryant R, Morony S, Stolina M, Kostenuik PJ, Dougall WC: Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis. Clin Exp Metastasis 2008, 25(2):119-129.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. H Hsu, D L Lacey, C R Dunstan, I Solovyev, A Colombero, E Timms, H L Tan, G Elliott, M J Kelley, I Sarosi, L Wang, X Z Xia, R Elliott, L Chiu, T Black, S Scully, C Capparelli, S Morony, G Shimamoto, M B Bass, W J Boyle, Proc Natl Acad Sci. 967Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, Tan HL, Elliott G, Kelley MJ, Sarosi I, Wang L, Xia XZ, Elliott R, Chiu L, Black T, Scully S, Capparelli C, Morony S, Shimamoto G, Bass MB, Boyle WJ: Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A 1999, 96(7):3540-3545.

Osteoprotegerin is a soluble decoy receptor or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand and can function as a paracrine survival factor for human myeloma cells. C M Shipman, P I Croucher, Cancer Res. 635Shipman CM, Croucher PI: Osteoprotegerin is a soluble decoy receptor or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand and can function as a paracrine survival factor for human myeloma cells. Cancer Res 2003, 63(5):912-916.

RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFalpha or anti-IL-1 therapies. M Stolina, G Schett, D Dwyer, S Vonderfecht, S Middleton, D Duryea, E Pacheco, G Van, B Bolon, U Feige, D Zack, P Kostenuik, Arthritis Res Ther. 116187Stolina M, Schett G, Dwyer D, Vonderfecht S, Middleton S, Duryea D, Pacheco E, Van G, Bolon B, Feige U, Zack D, Kostenuik P: RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFalpha or anti-IL-1 therapies. Arthritis Res Ther 2009, 11(6):R187.

Fully human therapeutic monoclonal antibodies. L M Weiner, J Immunotherapy. 291Weiner LM: Fully human therapeutic monoclonal antibodies. J Immunotherapy 2006, 29(1):1-9.

RANKL-RANK signaling in osteoclastogenesis and bone disease. T Wada, T Nakashima, N Hiroshi, J M Penninger, Trends Mol Med. 21Wada T, Nakashima T, Hiroshi N, Penninger JM: RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med 2006, 2(1):17-25.

Interaction with tumor necrosis factor receptor-associated factors and activation of NF-kappab and c-Jun N-terminal kinase. B G Darnay, V Haridas, J Ni, P A Moore, B B Aggarwal, J Biol Chem. 27332Characterization of the intracellular domain of receptor activator of NF-kappaB (RANK)Darnay BG, Haridas V, Ni J, Moore PA, Aggarwal BB: Characterization of the intracellular domain of receptor activator of NF-kappaB (RANK). Interaction with tumor necrosis factor receptor-associated factors and activation of NF-kappab and c-Jun N-terminal kinase. J Biol Chem 1998, 273(32):20551-20555.

Glutaredoxin2 isoform b (Glrx2b) promotes RANKL-induced osteoclastogenesis through activation of the p38-MAPK signaling pathway. J T Yeon, S W Choi, K I Park, M K Choi, J J Kim, B S Youn, M S Lee, J Oh, BMB Rep. 453Yeon JT, Choi SW, Park KI, Choi MK, Kim JJ, Youn BS, Lee MS, Oh J: Glutaredoxin2 isoform b (Glrx2b) promotes RANKL-induced osteoclastogenesis through activation of the p38-MAPK signaling pathway. BMB Rep 2012, 45(3):171-176.

Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. P Soriano, C Montgomery, R Geske, A Bradley, Cell. 644Soriano P, Montgomery C, Geske R, Bradley A: Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 1991, 64(4):693-702.

Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J G Emery, P Mcdonnell, M B Burke, K C Deen, S Lyn, C Silverman, E Dul, E R Appelbaum, C Eichman, R Diprinzio, R A Dodds, I E James, M Rosenberg, J C Lee, P R Young, J Biol Chem. 27323Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, Dul E, Appelbaum ER, Eichman C, DiPrinzio R, Dodds RA, James IE, Rosenberg M, Lee JC, Young PR: Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998, 273(23):14363-14367.

Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. F Ikeda, R Nishimura, T Matsubara, S Tanaka, J Inoue, S V Reddy, K Hata, K Yamashita, T Hiraga, T Watanabe, T Kukita, K Yoshioka, A Rao, T Yoneda, J Clin Invest. 1144Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata K, Yamashita K, Hiraga T, Watanabe T, Kukita T, Yoshioka K, Rao A, Yoneda T: Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest 2004, 114(4):475-484.

The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation. S E Lee, K M Woo, S Y Kim, H M Kim, K Kwack, Z H Lee, H H Kim, Bone. 301Lee SE, Woo KM, Kim SY, Kim HM, Kwack K, Lee ZH, Kim HH: The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation. Bone 2002, 30(1):71-77.

Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). M Matsumoto, T Sudo, T Saito, H Osada, M Tsujimoto, J Biol Chem. 27540Matsumoto M, Sudo T, Saito T, Osada H, Tsujimoto M: Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J Biol Chem 2000, 275(40):31155-31161.

T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. H Takayanagi, K Ogasawara, S Hida, T Chiba, S Murata, K Sato, A Takaoka, T Yokochi, H Oda, K Tanaka, K Nakamura, T Taniguchi, Nature. 4086812Takayanagi H, Ogasawara K, Hida S, Chiba T, Murata S, Sato K, Takaoka A, Yokochi T, Oda H, Tanaka K, Nakamura K, Taniguchi T: T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature 2000, 408(6812):600-605.

Suppressed severity of collagen-induced arthritis by in vivo transfection of nuclear factor kappaB decoy oligodeoxynucleotides as a gene therapy. T Tomita, E Takeuchi, N Tomita, R Morishita, M Kaneko, K Yamamoto, T Nakase, H Seki, K Kato, Y Kaneda, T Ochi, Arthritis Rheum. 4212Tomita T, Takeuchi E, Tomita N, Morishita R, Kaneko M, Yamamoto K, Nakase T, Seki H, Kato K, Kaneda Y, Ochi T: Suppressed severity of collagen-induced arthritis by in vivo transfection of nuclear factor kappaB decoy oligodeoxynucleotides as a gene therapy. Arthritis Rheum 1999, 42(12):2532-2542.

Role of RANKL in physiological and pathological bone resorption and therapeutics targeting the RANKL-RANK signaling system. S Tanaka, K Nakamura, N Takahasi, T Suda, Immunol Rev. 208Tanaka S, Nakamura K, Takahasi N, Suda T: Role of RANKL in physiological and pathological bone resorption and therapeutics targeting the RANKL-RANK signaling system. Immunol Rev 2005, 208:30-49.

Approaches for chemically synthesized siRNA and vector-mediated RNAi. M Amarzguioui, J J Rossi, D Kim, FEBS Lett. 57926Amarzguioui M, Rossi JJ, Kim D: Approaches for chemically synthesized siRNA and vector-mediated RNAi. FEBS Lett 2005, 579(26):5974-5981.

Delivery of RNA interference. C X Li, A Parker, E Menocal, S Xiang, L Borodyansky, J H Fruehauf, 10.1186/1471-2474-13-154Cell Cycle. 518Li CX, Parker A, Menocal E, Xiang S, Borodyansky L, Fruehauf JH: Delivery of RNA interference. Cell Cycle 2006, 5(18):2103-2109. doi:10.1186/1471-2474-13-154

Inhibition of osteoclastogenesis by RNA interference targeting RANK. Ma, BMC Musculoskeletal Disorders. 13154Cite this article asCite this article as: Ma et al.: Inhibition of osteoclastogenesis by RNA interference targeting RANK. BMC Musculoskeletal Disorders 2012 13:154.


Terminal chromosome 4q deletion syndrome in an infant with hearing impairment and moderate syndromic features: review of literature

Barbara Vona
Indrajit Nanda
Cordula Neuner
Jörg Schröder
Vera M Kalscheuer
Wafaa Shehata-Dieler
Thomas Haaf
Terminal chromosome 4q deletion syndrome in an infant with hearing impairment and moderate syndromic features: review of literature
C A S E R E P O R T Open Access
Background: Terminal deletions of chromosome 4q are associated with a broad spectrum of phenotypes including cardiac, craniofacial, digital, and cognitive impairment. The rarity of this syndrome renders genotype-phenotype correlation difficult, which is further complicated by the widely different phenotypes observed in patients sharing similar deletion intervals. Case presentation: Herein, we describe a boy with congenital hearing impairment and a variety of moderate syndromic features that prompted SNP array analysis disclosing a heterozygous 6.9 Mb deletion in the 4q35.1q35.2 region, which emerged de novo in the maternal germ line. Conclusion: In addition to the index patient, we review 35 cases from the literature and DECIPHER database to attempt genotype-phenotype correlations for a syndrome with great phenotypic variability. We delineate intervals with recurrent phenotypic overlap, particularly for cleft palate, congenital heart defect, intellectual disability, and autism spectrum disorder. Broad phenotypic presentation of the terminal 4q deletion syndrome is consistent with incomplete penetrance of the individual symptoms.

Background

Terminal deletions of chromosome 4q are a rare event with an approximate incidence of 1 in 100,000 [1,2]. While the majority are de novo cases, an estimated 10-20% are the unbalanced product of a parental reciprocal translocation. Furthermore, some pediatric cases with classical phenotypes have inherited their 4q deletion from a parent described as either normal or only mildly affected [3][4][5][6]. Although there is a high degree of phenotypic variation in those presenting overlapping deletion intervals, there is a general consensus that chromosome 4q deletion syndrome is characterized by intellectual disability (ID), craniofacial dysmorphism, rotated or low-set ears, cleft palate (CP), micrognathia, congenital heart defects (CHD), craniofacial, skeletal and digital abnormalities, and occasionally autism spectrum disorder (ASD), behavioural disorders, and developmental delay [7][8][9]. Chromosome 4q deletions are divided in two different subgroups depending on the region of 4q that is deleted: interstitial, spanning the centromere through 4q28.3 and terminal, from 4q31.1 to 4qter. Although both deletion types each have highly variable phenotypic associations, terminal deletion cases present a broader phenotypic range including CHD, craniofacial and skeletal abnormalities. The 4q33 region has been proposed as critical for ulnar deficiency, cleft lip and palate, and brain development [10].

Herein, we report on an eight year-old boy with moderate dysmorphic features and a de novo deletion in the 4q35.1q35.2 region. By analyzing the considerable phenotypic variability of terminal 4q deletion cases from the literature and DECIPHER database, we attempt to delineate critical intervals for common phenotypic features.

Case presentation

Clinical report

The proband is the only child of two healthy unrelated parents of German ethnicity, born at a gestational age of 38.3 weeks, after an uncomplicated pregnancy and normal spontaneous delivery. Birth weight was 3,125 g (25 th centile), APGAR scores of nine and ten at one and five minutes, respectively, cord blood pH was 7.3, and an unremarkable otoacoustic emissions newborn hearing screening test was recorded. At four months of age, he had bilateral hearing impairment in the 60 dB range and was fitted with hearing aids. We sequenced genes commonly screened for hearing loss, including GJB2 (MIM: 121011), GJB3 (MIM: 603324), and GJB6 (MIM: 604418). Sequencing disclosed a heterozygous mutation in GJB3 c.94C > T, p.Arg32Trp (rs1805063; minor allele frequency T = 0.015), which is a well-described autosomal recessive deafness gene requiring a second heterozygous mutation either in trans or in compound heterozygous configuration to convey hearing loss. A targeted deafness gene next generation sequencing panel was negative for other pathogenic mutations.

In the first year of life, he was diagnosed with aortic isthmus stenosis, corrected via balloon angioplasty, and a patent foramen ovale. He demonstrated shortened PQ intervals on an electrocardiogram indicative of an atrioventricular node irregularity. Regular pediatric cardiology follow-up was recommended. He also presented with chronic Eustachian tube dysfunction that was treated several times with myringotomy tubes, as well as a bifid uvula. In the fifth year of life, a submucous CP was detected. During the same year, he underwent corrective surgery for the CP and velopharyngeal insufficiency. Additionally, he presented with bilateral cryptorchidism that required testicular orchiopexie. An abdominal sonogram could not rule out the possibility of a left duplex kidney; urine analysis was within normal limits. Despite a small thyroid, he had normal thyroid function on lab testing. His blood profile was unremarkable apart from mild concurrent deficiencies of blood coagulation factors IX (56%), XI (48%) and XII (38%). He had an elevated prothrombin time of 46.5 s (normal: 25-39 s) and an elevated lupus anticoagulant confirmatory test of 1.26 (normal: 0.91-1.07). Further coagulation testing was negative for von Willebrand disease.

Psychological developmental evaluation at the age of three to four years showed mild general developmental delay (six months). Subsequent evaluations showed normal development. Neurological evaluation at the age five showed a lack of age-appropriate coordination. He also had delayed speech and language development, likely secondary, at least in part, to his hearing impairment and extensive hospitalization history. Currently, he attends regular school and does not require remedial classroom instruction.

Methods

Classical cytogenetic and fluorescence in situ hybridization (FISH) analyses

Chromosomes of the proband and his parents were prepared from peripheral blood lymphocyte cultures and analyzed by GTG-banding at the 500 band resolution. FISH was carried out using selected BAC probes from the deleted region. BAC DNA was labelled by nick translocation with fluorescein-12-dUTP (Roche Diagnostics, Mannheim, Germany) or tetramethyl-rhodamine-5-dUTP (Roche), and hybridized overnight to denatured chromosomes. Image acquisition and analysis were performed using FISHView 2.0 software (Applied Spectral Imaging, Edingen-Neckarhausen, Germany).

Copy number variation and genotype analyses

Genomic DNA (gDNA) of the proband and his parents was prepared from peripheral blood by standard salt extraction method. The Illumina Omni1-Quad v1.0 SNP array (Illumina, San Diego, CA, USA), with >1.1 million SNP markers, was used for whole genome genotyping and copy number variation (CNV) detection. 200 ng gDNA were utilized in an Illumina Infinium HD Ultra Assay according to the manufacturer's specifications. Data were analyzed using GenomeStudio (v2011.1) software with both cnvPartition 3.2.0 (Illumina) and Quan-tiSNP 2.2 copy number algorithm [11]. Genotypes of father, mother and proband were obtained from the SNP array for parent-of-origin determination. HaploPainter [12] was used in combination with manual intervention to illustrate the absence of maternal genotypes in the deletion patient. The terminal 4q monosomy was validated by real-time quantitative polymerase chain reaction (qPCR) of FRG1 exons 1, 8, and DUX4L6 using the Sen-siMix SYBR Green kit (Bioline, Luckenwalde, Germany).

Mapping critical intervals for terminal 4q deletion syndrome phenotypes

This study makes use of data generated by the DECIPHER consortium, which is funded by the Wellcome Trust ( [6][7][8][9][10][13][14][15][16][17][18][19][20][21][22], phenotypic and deletion overlaps among individuals with monosomies spanning different sizes were delineated. We used the UCSC Genome Browser Custom Track (http://genome-euro.ucsc. edu/cgi-bin/hgCustom) to map these cases and targeted the narrowest critical interval for CP, CHD, ID, and ASD.

Results

Classical and molecular cytogenetic analyses

Conventional chromosome banding analysis of the proband revealed a 46,XY karyotype without gross abnormalities. However, the distal G-band negative region in the long arm of chromosome 4 corresponding to q35.1q35.2 appeared to be somewhat smaller in one of the homologs, suggestive of a loss of chromosome material ( Figure 1A). Both parents had normal karyotypes without evidence of deletion on chromosome 4q.

To validate the deletion in the proband, SNP array analysis was performed which disclosed a 6.9 Mb heterozygous deletion on chromosome 4q35.1q35.2 (184,046,156-190,901,117 bp from rs17074417 to rs10005101, hg19) ( Figure 1B). qPCR analysis of FRG1 exons 1, 8, and DUX4L6 confirmed that the distal deletion breakpoint extends beyond 190,939,252 bp (data not shown), encompassing a total of 42 annotated genes (18 OMIM genes). Based on these results, the proband's karyotype could be assigned as 46,XY,del(4)(q35.1q35.2). SNP array analyses of maternal and paternal DNA did not indicate CNV for chromosome 4q in the parental karyotypes, consistent with a de novo deletion in the child. Informative SNPs from the terminal 4q region for which the mother and father have divergent genotypes revealed a loss of maternal genotypes in the child ( Figure 1C), compatible with maternal origin of the deleted chromosome.

FISH analysis was performed with BACs from the proximal flanking region 4q35.1 (RP11-188P17) and the deleted region 4q35.1q35.2 (RP11-775P18, RP11-118M15, and RP11-652J12). As expected, the flanking BAC probe hybridized to both chromosomes in the proband and parental metaphase spreads. Probes from the deleted region recognized only one chromosome 4 homolog of the patient, but were present on both chromosome 4q35.1q35.2 copies of father and mother ( Figure 1A). Obviously, the de novo deletion is not due to a cytogenetically cryptic subtelomeric translocation in a parental karyotype.

Genotype-phenotype correlation of terminal 4q deletion syndrome

We created a map of terminal 4q deletion syndrome cases through reviewing the literature and DECIPHER database. Figure 2 (upper section) presents 36 deletion cases (including our own) meeting our interval criteria and after controlling for normal CNV from the Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home). Although we had to estimate best fit intervals for cases describing deletions using various low-resolution methods, we were able to roughly map out five critical regions for four common 4q deletion syndrome phenotypes: CP, CHD, ID, and ASD. Figure 2 (middle section) and Additional file 1: Table S1 show the gene content of the deletion region, with an emphasis on genes implicated in the various associated phenotypes. Additional file 2: Table S2 details case summaries including approximate deletion sizes, inheritance and phenotype information.

A locus (chr. 4: 155,600,001-158,373,133 bp) for CP (Figure 2, purple) was mapped with two out of three cases spanning this region having CP [7,10]. The gene PDGFC is important for development of the palate with implication in non-syndromic orofacial clefting [23]. Furthermore, Figure 2 Genotype-phenotype correlation of terminal 4q deletions. The upper part of the figure presents the mapped deletion intervals in chromosome 4q31.1qter including the present case, marked with a box, and 35 additional cases from DECIPHER and the literature. The red bars delineate the deletion region for each case. Highlighted intervals indicate critical regions for common phenotypes among the cases. Depicted, from left to right, are the intervals for cleft palate (purple), congenital heart defect region 1 (light green), intellectual disability (dark green), congenital heart defect region 2 (red), and autism spectrum disorder (turquoise). Orange indicates the mapping interval of the DFNA24 locus. The middle section shows the gene content of the 4q31.1qter region. Likely disease-relevant genes overlapping with critical deletion intervals are boxed. The bottom diagram shows the deCODE recombination map, highlighting male and female recombination hotspots in the terminal 4q deletion syndrome region.

Pdgfc −/− knockout mice display clefting [24]. Thirteen cases under evaluation indicate CP, suggesting an additional critical interval involved in palate formation.

Congenital heart defects were mapped to two separate regions. The first region (Figure 2, light green) spans a large interval (chr. 4: 160,717,000-178,579,037 bp) unable to be further subdivided based on the cases presented. There are 17 cases with various cardiac phenotypes, 13 of which overlap with the proposed interval, with three individuals unique to this first CHD locus [10,18,21]. This interval contains two genes of interest (Additional file 1: Table S1). TLL1 is important for mammalian heart septation [25]. Mice with abnormalities in this gene die from blood circulation failure [26]. From mouse and zebrafish experiments, HAND2 is also involved in cardiac morphogenesis, angiogenesis, and formation of the right ventricle and aortic arch arteries and, interestingly, plays a role in limb formation [27,28]. Although many individuals presented digital and forearm deficiencies, we were not able to clearly map these phenotypes to this region as well.

The second CHD locus (chr. 4: 184,046,156-186,997, 806 bp) maps in a region containing 12 out of 17 overlapping cases with cardiac phenotypes (Figure 2, red), two of whom uniquely overlap with this region. The critical interval contains two adjacent genes, PDLIM3 and SORBS2, implicated in cardiac development. PDLIM3 is essential for right ventricular development and thought to enhance mechanical strength stability of cardiac muscle during mouse development [29]. SORBS2 is highly expressed in the intercalated disk in normal cardiac tissue [30]. Additionally, SORBS2 could have implication in CP formation, since case #20 [9] with CP has a small deletion (chr. 4: 186,533,075-186,997,806 bp) exclusively affecting SORBS2 and TLR3 (Figure 2, upper and middle section). Ten out of 13 total individuals with CP overlapped with this region, but the proximal border was too large to map an informative locus.

A smaller region (chr. 4: 171,144,641-175,897,427 bp) within the first CHD interval may account for ID, with eight of 15 individuals having ID (Figure 2, dark green). While no gene is presently linked to ID in this region, the gene SCRG1 is highly expressed in the brain and has differential regulation in schizophrenia and bipolar disorder [31] (Additional file 1: Table S1). Lack of genomic variation among healthy individuals in the Database of Genomic Variants and strong evolutionary conservation (data not shown) further emphasize the importance of normal copy number of this ID region.

A number of reports implicate chromosome 4q35.2 in ASD [20,22]. While only four cases reviewed here have ASD (Additional file 2: Table S2), all four overlap one narrow interval (chr. 4: 187,234,067-188,424,639 bp) (Figure 2, turquoise) with only three genes (MTNR1A, FAT1 and F11), that was first reported in a boy with ASD [22]. FAT1 has been associated with bipolar affective disorder [32] and ASD [33], and is essential for controlling developmental cell proliferation [34].

Mild factor XI deficiency and elevated prothrombin time in our proband are presumably explained through deletion of F11 [35,36] and an adjacent coagulation gene, KLKB1 [37]. Surprisingly, the mild bleeding tendencies that can be associated with F11 and KLKB1 deletions have not been discussed in great detail yet, although many children with terminal 4q deletion syndrome require multiple surgeries.

The first clinical symptom of our patient with a 6.9 Mb deletion (chr. 4: 184,046,156-190,901,117 bp) was mild to severe bilateral hearing loss. Two additional cases with larger deletions, DECIPHER case 256186 and the case from Calabrese et al., 1997 [16], also reported hearing impairment. In a Swiss-German kindred with autosomal dominant non-syndromic hearing loss, an autosomal dominant deafness locus, DFNA24 (MIM: 606282) was mapped to an 8.1 Mb region (chr. 4: 183,200,000-191,154,276 bp) (Figure 2, orange) on chromosome 4q35qter [38,39]. However, in this context it is important to emphasize that 11 normal hearing terminal 4q deletion cases overlap completely and nine normal hearing cases overlap partially with the DFNA24 interval. Thus, loss of one locus copy is not sufficient to cause DFNA24. A cumulative effect of rare, pathogenic variants in different deafness genes scattered across the genome (i.e. haploinsufficiency for DFNA24) could contribute to hearing impairment [40].

Mouse knockout experiments suggest that Casp3, which is contained in the critical region, is required for proper functioning of the cochlea [41][42][43]. Casp3 −/− mice indicated sensorineural hearing loss, whereas Casp3 +/− mice displayed intermediate vestibular dysfunction, as well as marginally increased hair cell counts.

Discussion

Since its first description [44], the genotype-phenotype delineation of chromosome 4q deletion syndrome has been complicated by extensive inconsistencies reported among individuals with similar deletion intervals. With >170 genes residing in the terminal 4q region, delineation of the phenotypes associated with such deletions presents a tremendous task toward understanding the complete spectral presentation of a syndrome with excessive phenotypic variability. The patient we present was analyzed with a high resolution SNP array to delineate the deletion interval and the parental origin of the de novo rearrangement. We found it especially challenging to finely map disease-relevant intervals with the various low-resolution techniques that used GTG banding [7,10,[13][14][15], FISH [16], and the different resolution arrays, including BAC aCGH [18], 1 Mb aCGH [19], 44 K aCGH [6,17,20], 105 K aCGH [9,22], and 300 K SNP array [21]. Another limitation includes possible variations in the depth of clinical descriptions listed, especially those from the DECIPHER database, which were not as detailed as the published cases. Collectively, casesupported critical regions for several distinct phenotypes such as CP, CHD, ID, and ASD were defined. In this context, it is important to emphasize that most phenotypic features that are associated with terminal 4q deletion syndrome show incomplete penetrance and/or are rather unspecific, which renders genotype-phenotype correlations difficult.

The overwhelming majority of cases are de novo possibly due to errors during meiotic recombination leading to a loss of chromosomal material from one parental allele. Meiotic crossovers preferentially occur at nonrandom hotspots which have been mapped according to frequency and spatial distribution in both males and females [45]. The deCODE recombination map [46] of the 4q31.1qter region illustrates an enrichment of both male and female hotspots along the major part of this interval ( Figure 2, bottom section). However, it is also possible that deletions arise in mitotically dividing spermatogonial and oogonial stem cells, respectively. The resulting germ-cell mosaicism would increase the likelihood of having another child with the same deletion.

In summary, the case presented here is the first to use a SNP array to determine the parent-of-origin of the large deletion. Assuming that the same gene(s) is underlying hearing impairment in terminal 4q deletion and DFNA24 patients, it may help further narrow the DFNA24 locus. Our case, in combination with the cases described in the literature and DECIPHER, accommodate a proposal of critical phenotypic intervals with possible genes of interest. This review is not intended as a holistic description of terminal chromosome 4q deletion syndrome. However, the ongoing reporting of precisely defined deletion intervals with higher resolution technologies will support eventual refinement and possible clarification of the genes and pathways responsible for the broad phenotypic presentation of deletions in this interval of chromosome 4q.

Consent

The study was approved by the Ethics Committee of the University of Würzburg. Full informed parental consent was obtained prior to initiating our investigation.

Additional files

Additional file 1: Table S1. Summary of disease-relevant genes in the deletion region with functions, phenotypes and cases with agreeable phenotypes.

Additional file 2: Table S2. Summary of our proband and cases from DECIPHER and the literature with deletions exclusively residing in the 4q31.1qter region.

Competing interest

No potential competing interest as well as commercial interests were disclosed.

Authors' contributions BV, IN and CN carried out the molecular cytogenetic and data analyses. JS and WSD performed a clinical analysis of the patient. VK provided materials. BV and TH researched the literature and wrote the manuscript. All authors have critically reviewed and approved the manuscript.

Figure 1
1Molecular karyotyping of the patient and his parents. (A) GTG-banding and FISH analysis of homologous chromosomes 4 in proband, mother and father. The chromosomes are counterstained with DAPI (blue). The proximal flanking BAC RP11-188P17 is labeled with fluorescein-dUTP (green) and the deleted BAC RP11-775P18 with rhodamine-dUTP (red). An arrowhead indicates the critical band q35.1q35.2 on the patient's derivative chromosome. (B) Illumina SNP array analysis (B allele frequency and log R ratio) of the 4q35.1q35.2 region in the boy with terminal 4q deletion syndrome. (C) Selected genotypes in the deletion interval from the Illumina array are depicted for proband (left), mother (middle), and father (right). Mendelian transmission errors (absence of maternal genotypes) in the proband are indicated in gray.

this article as: Vona et al.: Terminal chromosome 4q deletion syndrome in an infant with hearing impairment and moderate syndromic features: review of literature. BMC Medical Genetics 2014 15:72.
http://www.biomedcentral.com/1471-2350/15/72
AcknowledgementsThe authors are grateful for the participation of the family for their willingness to engage in this study. We also thank Prof. Dr. Holger Thiele from the Cologne Center for Genomics for constructive dialogue about HaploPainter, Dr. Christopher Riley at Phoenix Children's Hospital for clinical assessment, and Andrea Hörning for assistance with karyotype analysis. This work was supported by the German Research Foundation (grant no. HA 1374/7-2) and the University of Würzburg in the funding programme Open Access Publishing.
The 4q-syndrome. E M Strehle, O A Ahmed, M Hameed, A Russell, Genet Couns. 12Strehle EM, Ahmed OA, Hameed M, Russell A: The 4q-syndrome. Genet Couns 2001, 12:327-339.

The phenotype of patients with 4q-syndrome. E M Strehle, H M Bantock, Genet Couns. 14Strehle EM, Bantock HM: The phenotype of patients with 4q-syndrome. Genet Couns 2003, 14:195-205.

Terminal deletion of the long arm of chromosome 4 in a mother and two sons. M Descartes, K Keppler-Noreuil, J Knops, J W Longshore, W H Finley, A J Carroll, Clin Genet. 50Descartes M, Keppler-Noreuil K, Knops J, Longshore JW, Finley WH, Carroll AJ: Terminal deletion of the long arm of chromosome 4 in a mother and two sons. Clin Genet 1996, 50:538-540.

Subtelomere FISH analysis of 11 688 cases: an evaluation of the frequency and pattern of subtelomere rearrangements in individuals with developmental disabilities. J B Ravnan, J H Tepperberg, P Papenhausen, A N Lamb, J Hedrick, D Eash, D H Ledbetter, C L Martin, J Med Genet. 43Ravnan JB, Tepperberg JH, Papenhausen P, Lamb AN, Hedrick J, Eash D, Ledbetter DH, Martin CL: Subtelomere FISH analysis of 11 688 cases: an evaluation of the frequency and pattern of subtelomere rearrangements in individuals with developmental disabilities. J Med Genet 2006, 43:478-489.

I Balikova, B Menten, T De Ravel, Le Caignec, C Thienpont, B Urbina, M Doco-Fenzy, M De Rademaeker, M Mortier, G Kooy, F Van Den Ende, J Devriendt, K Fryns, J P Speleman, F Vermeesch, J R , Subtelomeric imbalances in phenotypically normal individuals. 28Balikova I, Menten B, de Ravel T, Le Caignec C, Thienpont B, Urbina M, Doco-Fenzy M, de Rademaeker M, Mortier G, Kooy F, van den Ende J, Devriendt K, Fryns JP, Speleman F, Vermeesch JR: Subtelomeric imbalances in phenotypically normal individuals. Hum Mutat 2007, 28:958-967.

Clinical and genomic characterization of distal duplications and deletions of chromosome 4q: study of two cases and review of the literature. M R Rossi, M S Dimaio, B Xiang, K Lu, H Kaymakcalan, M Seashore, M J Mahoney, P Li, Am J Med Genet A. 149Rossi MR, DiMaio MS, Xiang B, Lu K, Kaymakcalan H, Seashore M, Mahoney MJ, Li P: Clinical and genomic characterization of distal duplications and deletions of chromosome 4q: study of two cases and review of the literature. Am J Med Genet A 2009, 149A:2788-2794.

Hypercalciuria and kidney calcifications in terminal 4q deletion syndrome: further evidence for a putative gene on 4q. M Giuffrè, La Placa, S Carta, M Cataliotti, A Marino, M Piccione, M Pusateri, F Meli, F Corsello, G , Am J Med Genet A. 126Giuffrè M, La Placa S, Carta M, Cataliotti A, Marino M, Piccione M, Pusateri F, Meli F, Corsello G: Hypercalciuria and kidney calcifications in terminal 4q deletion syndrome: further evidence for a putative gene on 4q. Am J Med Genet A 2004, 126A:186-190.

The spectrum of 4q-syndrome illustrated by a case series. E M Strehle, D Gruszfeld, D Schenk, S G Mehta, I Simonic, T Huang, Gene. 506Strehle EM, Gruszfeld D, Schenk D, Mehta SG, Simonic I, Huang T: The spectrum of 4q-syndrome illustrated by a case series. Gene 2012, 506:387-391.

. E M Strehle, L Yu, J A Rosenfeld, S Donkervoort, Y Zhou, T J Chen, J E Martinez, Y S Fan, D Barbouth, H Zhu, A Vaglio, R Smith, C A Stevens, C J Curry, Strehle EM, Yu L, Rosenfeld JA, Donkervoort S, Zhou Y, Chen TJ, Martinez JE, Fan YS, Barbouth D, Zhu H, Vaglio A, Smith R, Stevens CA, Curry CJ,

Genotype-phenotype analysis of 4q deletion syndrome: proposal of a critical region. R L Ladda, Z J Fan, J E Fox, J A Martin, Abdel-Hamid Hz, E A Mccracken, B C Mcgillivray, D Masser-Frye, T Huang, Am J Med Genet A. 158Ladda RL, Fan ZJ, Fox JE, Martin JA, Abdel-Hamid HZ, McCracken EA, McGil- livray BC, Masser-Frye D, Huang T: Genotype-phenotype analysis of 4q de- letion syndrome: proposal of a critical region. Am J Med Genet A 2012, 158A:2139-2151.

Interstitial deletion 4q32-34 with ulnar deficiency: 4q33 may be the critical region in 4q terminal deletion syndrome. S L Keeling, L Lee-Jones, P Thompson, Am J Med Genet. 99Keeling SL, Lee-Jones L, Thompson P: Interstitial deletion 4q32-34 with ulnar deficiency: 4q33 may be the critical region in 4q terminal deletion syndrome. Am J Med Genet 2001, 99:94-98.

QuantiSNP: an objective Bayes hidden-Markov model to detect and accurately map copy number variation using SNP genotyping data. S Colella, C Yau, J M Taylor, G Mirza, H Butler, P Clouston, A S Bassett, A Seller, C C Holmes, J Ragoussis, Nucleic Acids Res. 35Colella S, Yau C, Taylor JM, Mirza G, Butler H, Clouston P, Bassett AS, Seller A, Holmes CC, Ragoussis J: QuantiSNP: an objective Bayes hidden-Markov model to detect and accurately map copy number variation using SNP genotyping data. Nucleic Acids Res 2007, 35:2013-2025.

HaploPainter: a tool for drawing pedigrees with complex haplotypes. H Thiele, P Nürnberg, Bioinformatics. 21Thiele H, Nürnberg P: HaploPainter: a tool for drawing pedigrees with complex haplotypes. Bioinformatics 2005, 21:1730-1732.

A terminal deletion of the long arm of chromosome 4 [46, XX, del(4)(q33)] in an infant with phenotypic features of Williams syndrome. R D Jefferson, J Burn, K L Gaunt, S Hunter, E V Davison, J Med Genet. 23Jefferson RD, Burn J, Gaunt KL, Hunter S, Davison EV: A terminal deletion of the long arm of chromosome 4 [46, XX, del(4)(q33)] in an infant with phenotypic features of Williams syndrome. J Med Genet 1986, 23:474-477.

Robin sequence and a deficiency of the left forearm in a girl with a deletion of chromosome 4q33-qter. F H Menko, K Madan, J A Baart, H L Beukenhorst, Am J Med Genet. 44Menko FH, Madan K, Baart JA, Beukenhorst HL: Robin sequence and a deficiency of the left forearm in a girl with a deletion of chromosome 4q33-qter. Am J Med Genet 1992, 44:696-698.

Deletion (4)(q33-> qter): a case report and review of the literature. Z Borochowitz, S A Shalev, I Yehudai, H Bar-El, H Dar, E Tirosh, J Child Neurol. 12Borochowitz Z, Shalev SA, Yehudai I, Bar-el H, Dar H, Tirosh E: Deletion (4)(q33-> qter): a case report and review of the literature. J Child Neurol 1997, 12:335-337.

Two newborns with chromosome 4 imbalances: deletion 4q33→q35 and ring r(4)(pterq35.2-qter). G Calabrese, A Giannotti, R Mingarelli, Di Gilio, M C Piemontese, M R Palka, G , Clin Genet. 51Calabrese G, Giannotti A, Mingarelli R, Di Gilio MC, Piemontese MR, Palka G: Two newborns with chromosome 4 imbalances: deletion 4q33→q35 and ring r(4)(pterq35.2-qter). Clin Genet 1997, 51:264-267.

A girl with del(4)(q33) and occipital encephalocele: clinical description and molecular genetic characterization of a rare patient. R Quadrelli, E M Strehle, A Vaglio, M Larrandaburu, B Mechoso, A Quadrelli, Y S Fan, T Huang, Genet Test. 11Quadrelli R, Strehle EM, Vaglio A, Larrandaburu M, Mechoso B, Quadrelli A, Fan YS, Huang T: A girl with del(4)(q33) and occipital encephalocele: clinical description and molecular genetic characterization of a rare patient. Genet Test 2007, 11:4-10.

Investigation of 4q-deletion in two unrelated patients using array CGH. S S Kaalund, R S Møller, A Teszas, M Miranda, G Kosztolanyi, R Ullmann, N Tommerup, Z Tümer, Am J Med Genet A. 146Kaalund SS, Møller RS, Teszas A, Miranda M, Kosztolanyi G, Ullmann R, Tommerup N, Tümer Z: Investigation of 4q-deletion in two unrelated patients using array CGH. Am J Med Genet A 2008, 146A:2431-2434.

Distal del(4) (q33) syndrome: detailed clinical presentation and molecular description with array-CGH. S Kitsiou-Tzeli, C Sismani, G Koumbaris, M Ioannides, E Kanavakis, A Kolialexi, A Mavrou, V Touliatou, P C Patsalis, Eur J Med Genet. 51Kitsiou-Tzeli S, Sismani C, Koumbaris G, Ioannides M, Kanavakis E, Kolialexi A, Mavrou A, Touliatou V, Patsalis PC: Distal del(4) (q33) syndrome: detailed clinical presentation and molecular description with array-CGH. Eur J Med Genet 2008, 51:61-67.

Identification and molecular characterization of two novel chromosomal deletions associated with autism. W H Chien, S S Gau, Y Y Wu, Y S Huang, J S Fang, Y J Chen, W T Soong, Y N Chiu, C H Chen, Clin Genet. 78Chien WH, Gau SS, Wu YY, Huang YS, Fang JS, Chen YJ, Soong WT, Chiu YN, Chen CH: Identification and molecular characterization of two novel chromosomal deletions associated with autism. Clin Genet 2010, 78:449-456.

Chromosome 4q deletion syndrome: narrowing the cardiovascular critical region to 4q32.2-q34.3. W Xu, A Ahmad, S Dagenais, R K Iyer, J W Innis, Am J Med Genet A. 158Xu W, Ahmad A, Dagenais S, Iyer RK, Innis JW: Chromosome 4q deletion syndrome: narrowing the cardiovascular critical region to 4q32.2-q34.3. Am J Med Genet A 2012, 158A:635-640.

12-year-old boy with a 4q35.2 microdeletion and involvement of MTNR1A, FAT1, and F11 genes. E L Youngs, R S Henkhaus, J A Hellings, M G Butler, Clin Dysmorphol. 21Youngs EL, Henkhaus RS, Hellings JA, Butler MG: 12-year-old boy with a 4q35.2 microdeletion and involvement of MTNR1A, FAT1, and F11 genes. Clin Dysmorphol 2012, 21:93-96.

Maternal transmission effect of a PDGF-C SNP on nonsyndromic cleft lip with or without palate from a Chinese population. D Wu, M Wang, X Wang, N Yin, T Song, H Li, F Zhang, Y Zhang, Z Ye, J Yu, D M Wang, Z Zhao, PLoS One. 746477Wu D, Wang M, Wang X, Yin N, Song T, Li H, Zhang F, Zhang Y, Ye Z, Yu J, Wang DM, Zhao Z: Maternal transmission effect of a PDGF-C SNP on nonsyndromic cleft lip with or without palate from a Chinese population. PLoS One 2012, 7:e46477.

A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling. H Ding, X Wu, H Bostrom, I Kim, N Wong, B Tsoi, M O'rourke, G Y Koh, P Soriano, C Betsholtz, T C Hart, M L Marazita, L L Field, P P Tam, A Nagy, Nat Genet. 36Ding H, Wu X, Bostrom H, Kim I, Wong N, Tsoi B, O'Rourke M, Koh GY, Soriano P, Betsholtz C, Hart TC, Marazita ML, Field LL, Tam PP, Nagy A: A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling. Nat Genet 2004, 36:1111-1116.

Mutations in mammalian tolloid-like 1 gene detected in adult patients with ASD. P Stańczak, J Witecka, A Szydło, E Gutmajster, M Lisik, A Auguściak-Duma, M Tarnowski, T Czekaj, H Czekaj, A L Sieroń, Eur J Hum Genet. 17Stańczak P, Witecka J, Szydło A, Gutmajster E, Lisik M, Auguściak-Duma A, Tarnowski M, Czekaj T, Czekaj H, Sieroń AL: Mutations in mammalian tolloid-like 1 gene detected in adult patients with ASD. Eur J Hum Genet 2009, 17:344-351.

The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart. T G Clark, S J Conway, I C Scott, P A Labosky, G Winnier, J Bundy, B L Hogan, D S Greenspan, Development. 126Clark TG, Conway SJ, Scott IC, Labosky PA, Winnier G, Bundy J, Hogan BL, Greenspan DS: The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart. Development 1999, 126:2631-2642.

Edn1 and hand2 interact in early regulation of pharyngeal arch outgrowth during zebrafish development. M M Sasaki, J T Nichols, C B Kimmel, PLoS One. 867522Sasaki MM, Nichols JT, Kimmel CB: Edn1 and hand2 interact in early regulation of pharyngeal arch outgrowth during zebrafish development. PLoS One 2013, 8:e67522.

Overdosage of Hand2 causes limb and heart defects in the human chromosomal disorder partial trisomy distal 4q. M Tamura, M Hosoya, M Fujita, T Iida, T Amano, A Maeno, T Kataoka, T Otsuka, S Tanaka, S Tomizawa, T Shiroishi, Hum Mol Genet. 22Tamura M, Hosoya M, Fujita M, Iida T, Amano T, Maeno A, Kataoka T, Otsuka T, Tanaka S, Tomizawa S, Shiroishi T: Overdosage of Hand2 causes limb and heart defects in the human chromosomal disorder partial trisomy distal 4q. Hum Mol Genet 2013, 22:2471-2481.

Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy. M Pashmforoush, P Pomies, K L Peterson, S Kubalak, J RossJr, A Hefti, U Aebi, M C Beckerle, K R Chien, Nat Med. 7Pashmforoush M, Pomies P, Peterson KL, Kubalak S, Ross J Jr, Hefti A, Aebi U, Beckerle MC, Chien KR: Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy. Nat Med 2001, 7:591-597.

Sorbin and SH3 domain-containing protein 2 is released from infarcted heart in the very early phase: proteomic analysis of cardiac tissues from patients. Y Kakimoto, S Ito, H Abiru, H Kotani, M Ozeki, K Tamaki, T Tsuruyama, J Am Heart Assoc. 2565Kakimoto Y, Ito S, Abiru H, Kotani H, Ozeki M, Tamaki K, Tsuruyama T: Sorbin and SH3 domain-containing protein 2 is released from infarcted heart in the very early phase: proteomic analysis of cardiac tissues from patients. J Am Heart Assoc 2013, 2:e000565.

Genome scans and gene expression microarrays converge to identify gene regulatory loci relevant in schizophrenia. M P Vawter, M E Atz, B L Rollins, K M Cooper-Casey, L Shao, W F Byerley, Hum Genet. 119Vawter MP, Atz ME, Rollins BL, Cooper-Casey KM, Shao L, Byerley WF: Genome scans and gene expression microarrays converge to identify gene regulatory loci relevant in schizophrenia. Hum Genet 2006, 119:558-570.

Genetic variation of the FAT gene at 4q35 is associated with bipolar affective disorder. R Abou Jamra, T Becker, A Georgi, T Feulner, J Schumacher, J Stromaier, F Schirmbeck, T G Schulze, P Propping, M Rietschel, M M Nöthen, S Cichon, Mol Psychiatry. 13Abou Jamra R, Becker T, Georgi A, Feulner T, Schumacher J, Stromaier J, Schirmbeck F, Schulze TG, Propping P, Rietschel M, Nöthen MM, Cichon S: Genetic variation of the FAT gene at 4q35 is associated with bipolar affective disorder. Mol Psychiatry 2008, 13:277-284.

Chromosomal microarray analysis of consecutive individuals with autism spectrum disorders or learning disability presenting for genetic services. J L Roberts, K Hovanes, M Dasouki, A M Manzardo, M G Butler, Gene. 535Roberts JL, Hovanes K, Dasouki M, Manzardo AM, Butler MG: Chromosomal microarray analysis of consecutive individuals with autism spectrum disorders or learning disability presenting for genetic services. Gene 2014, 535:70-78.

Angiotensin II induces Fat1 expression/ activation and vascular smooth muscle cell migration via Nox1-dependent reactive oxygen species generation. T Bruder-Nascimento, P Chinnasamy, D F Riascos-Bernal, S B Cau, G E Callera, R M Touyz, R C Tostes, N E Sibinga, J Mol Cell Cardiol. 66Bruder-Nascimento T, Chinnasamy P, Riascos-Bernal DF, Cau SB, Callera GE, Touyz RM, Tostes RC, Sibinga NE: Angiotensin II induces Fat1 expression/ activation and vascular smooth muscle cell migration via Nox1-dependent reactive oxygen species generation. J Mol Cell Cardiol 2013, 66:18-26.

An Alu-mediated 31.5-kb deletion as the cause of factor XI deficiency in 2 unrelated patients. M Mitchell, L Dai, G Savidge, A Alhaq, Blood. 104Mitchell M, Dai L, Savidge G, Alhaq A: An Alu-mediated 31.5-kb deletion as the cause of factor XI deficiency in 2 unrelated patients. Blood 2004, 104:2394-2396.

Congenital factor XI deficiency: an update. S Duga, O Salomon, Semin Thromb Hemost. 39Duga S, Salomon O: Congenital factor XI deficiency: an update. Semin Thromb Hemost 2013, 39:621-631.

Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms. V L Koumandou, A Scorilas, PLoS One. 868074Koumandou VL, Scorilas A: Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms. PLoS One 2013, 8:e68074.

A novel locus (DFNA24) for prelingual nonprogressive autosomal dominant nonsyndromic hearing loss maps to 4q35-qter in a large Swiss German kindred. F M Häfner, A A Salam, T E Linder, D Balmer, A Baumer, A A Schinzel, T Spillmann, S M Leal, Am J Hum Genet. 66Häfner FM, Salam AA, Linder TE, Balmer D, Baumer A, Schinzel AA, Spillmann T, Leal SM: A novel locus (DFNA24) for prelingual nonprogressive autosomal dominant nonsyndromic hearing loss maps to 4q35-qter in a large Swiss German kindred. Am J Hum Genet 2000, 66:1437-1442.

Phenotypic characterization of DFNA24: prelingual progressive sensorineural hearing impairment. R L Santos, F M Häfner, P L Huygen, T E Linder, A A Schinzel, T Spillmann, S M Leal, Audiol Neurootol. 11Santos RL, Häfner FM, Huygen PL, Linder TE, Schinzel AA, Spillmann T, Leal SM: Phenotypic characterization of DFNA24: prelingual progressive sensorineural hearing impairment. Audiol Neurootol 2006, 11:269-275.

Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations. B Vona, T Müller, I Nanda, C Neuner, Mah Hofrichter, J Schröder, O Bartsch, A Läßig, A Keilmann, S Schraven, F Kraus, W Shehata-Dieler, T Haaf, Vona B, Müller T, Nanda I, Neuner C, Hofrichter MAH, Schröder J, Bartsch O, Läßig A, Keilmann A, Schraven S, Kraus F, Shehata-Dieler W, Haaf T: Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations.

. 24875298Genet Med. Genet Med 2014. PMID 24875298:Epub ahead of print.

Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice. H Morishita, T Makishima, C Kaneko, Y S Lee, N Segil, K Takahashi, A Kuraoka, T Nakagawa, J Nabekura, K Nakayama, K I Nakayama, Biochem Biophys Res Comm. 284Morishita H, Makishima T, Kaneko C, Lee YS, Segil N, Takahashi K, Kuraoka A, Nakagawa T, Nabekura J, Nakayama K, Nakayama KI: Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice. Biochem Biophys Res Comm 2001, 284:142-149.

Melody, an ENU mutation in caspase 3, alters the catalytic cysteine residue and causes sensorineural hearing loss in mice. A Parker, R E Hardisty-Hughes, L Wisby, S Joyce, S D Brown, Mamm Genome. 21Parker A, Hardisty-Hughes RE, Wisby L, Joyce S, Brown SD: Melody, an ENU mutation in caspase 3, alters the catalytic cysteine residue and causes sensorineural hearing loss in mice. Mamm Genome 2010, 21:565-576.

Inner ear dysfunction in caspase-3 deficient mice. T Makishima, L Hochman, P Armstrong, E Rosenberger, R Ridley, M Woo, A Perachio, S Wood, BMC Neurosci. 12102Makishima T, Hochman L, Armstrong P, Rosenberger E, Ridley R, Woo M, Perachio A, Wood S: Inner ear dysfunction in caspase-3 deficient mice. BMC Neurosci 2011, 12:102.

A large deletion of the long arm of chromosome no. 4 in a child with limb abnormalities. C H Ockey, G V Feldman, M E Macaulay, M J Delaney, Arch Dis Child. 42Ockey CH, Feldman GV, Macaulay ME, Delaney MJ: A large deletion of the long arm of chromosome no. 4 in a child with limb abnormalities. Arch Dis Child 1967, 42:428-434.

Where the crossovers are: recombination distributions in mammals. L Kauppi, A J Jeffreys, S Keeney, Nat Rev Genet. 5Kauppi L, Jeffreys AJ, Keeney S: Where the crossovers are: recombination distributions in mammals. Nat Rev Genet 2004, 5:413-424.

Fine-scale recombination rate differences between sexes, populations and individuals. A Kong, G Thorleifsson, D F Gudbjartsson, G Masson, A Sigurdsson, A Jonasdottir, G B Walters, A Jonasdottir, A Gylfason, K T Kristinsson, S A Gudjonsson, M L Frigge, A Helgason, U Thorsteinsdottir, K Stefansson, Nature. 467Kong A, Thorleifsson G, Gudbjartsson DF, Masson G, Sigurdsson A, Jonasdottir A, Walters GB, Jonasdottir A, Gylfason A, Kristinsson KT, Gudjonsson SA, Frigge ML, Helgason A, Thorsteinsdottir U, Stefansson K: Fine-scale recombination rate differences between sexes, populations and individuals. Nature 2010, 467:1099-1103.