CHARGE syndrome is an autosomal dominant disorder caused in about two-third of cases by mutations in the CHD7 gene. For other genetic diseases e.g. hereditary spastic paraplegia, it was shown that interacting partners are involved in the underlying cause of the disease. These data encouraged us to search for CHD7 binding partners by a yeast two-hybrid library screen and CHD8 was identified as an interacting partner. The result was confirmed by a direct yeast two-hybrid analysis, co-immunoprecipitation studies and by a bimolecular fluorescence complementation assay. To investigate the function of CHD7 missense mutations in the CHD7-CHD8 interacting area on the binding capacity of both proteins, we included three known missense mutations (p.His2096Arg, p.Val2102Ile and p.Gly2108Arg) and one newly identified missense mutation (p.Trp2091Arg) in the CHD7 gene and performed both direct yeast two-hybrid and co-immunoprecipitation studies. In the direct yeast two-hybrid system, the CHD7-CHD8 interaction was disrupted by the missense mutations p.Trp2091Arg, p.His2096Arg and p.Gly2108Arg, whereas in the co-immunoprecipitation studies disruption of the CHD7-CHD8 interaction by the mutations could not be observed. The results lead to the hypothesis that CHD7 and CHD8 proteins are interacting directly and indirectly via additional linker proteins. Disruption of the direct CHD7-CHD8 interaction might change the conformation of a putative large CHD7-CHD8 complex and could be a disease mechanism in CHARGE syndrome.
BackgroundMutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry.Principle findingsThe hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues.ConclusionOur results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders.
Studying the genetic diversity of Mongolian Argali sheep populations using microsatellite loci requires an accurate and world-standard method than Polyacrylamide gel electrophoresis (PAGE). Therefore, in this study, we developed a multiplex PCR for 3 high polymorphism loci (BM302, INRA040, BM4505) using fluorescent labeled primers (FAM, HEX, TAMRA), and tested the usability of this methodology to simultaneously amplify the alleles of multiple micro[1]satellite loci in a short amount of time. Using our multiplex PCR method, the alleles of those 3 loci were successfully amplified on a total of 99 samples from the Altai, Gobi, Khangai, and Khoridol Saridag populations, and their allele lengths were determined by the fragment analysis method. General genetic parameters were evaluated to determine the observed length of alleles on each microsatellite locus that was suitable for research. The number of alleles (Na) observed in Altai (15.00), Gobi Desert (16.67), and Khangai (13.33) populations were relatively high, while in the Khoridol Saridag population, it was lower (9.00). The number of effective alleles (Ne) was comparatively low in Altai (6.73), Gobi Desert (8.02), and Khoridol Saridag (4.2) populations, but considerably uniform in the Khangai population (9.94). Moreover, observed (Ho) and expected heterozygosity (He) was highest in Altai (0.80, 0.84), Gobi (0.74, 0.87), and Khangai (0.85, 0.89) populations. Although the genetic diversity of the BM302, BM4505, and INRA040 loci was high, the majority of the detected alleles had low frequency. In conclusion, multiplex PCR was successfully optimized and the amplified fragments were analyzed which resulted in preliminary population genetic results in a short period. Future research uti[1]lizing this multiplex PCR and fragment analysis methodology should be conducted with more microsatellite markers, leading to precise results concerning the conservation genetics of Argali sheep. Монгол орны аргаль хонины популяцуудын микросателлитын судалгаанд мультиплекс ПГУ ба фрагментийн анализын аргазүйг тогтворжуулан туршсан дүнгээс Монгол орны аргаль хонины генетик олон янз байдлыг микросателлитын локусуудаар судлахад ПААГ-аас илүү өндөр нарийвчлалтай, дэлхийн стандартад нийцсэн фрагмент анализын аргазүйгээр хийх шаардлага тулгарсан. Ингэхдээ олон микросателлитын локусын аллелийг богино хугацаанд олшруулахын тулд бид флуоресценц 3 өөр өнгөтэй (FAM, HEX, TAMRA) бодисоор тэмдэглэсэн праймераар полиморфизм өндөртэй 3 локусыг (BM302, INRA040, BM4505) сонгон мультиплекс ПГУ-ын арга зүй боловсруулж, энэ аргазүйг цаашид ашиглах боломжийг турших зорилгоор энэ судалгааг хийв. Боловсруулсан мултиплекс ПГУ-ын арга зүйг ашиглан Алтай, Говь, Хангай, Хорьдол Сарьдагийн популяцуудын нийт 99 дээжид дээрх 3 локусын аллелиудыг амжилттай олшруулж, аллелийн уртыг тодорхойлсон. Түүнчлэн микросателлитын локус бүрийн аллелиудын урт нь судалгаанд ашиглахад тохиромжтой эсэхийг шалган ерөнхий генетик үзүүлэлтүүдийг тооцоход ажиглагдсан аллелийн тоо Алтай (15.00), Говь (16.67), Хангайн (13.33) популяцад их, Хорьдол Сарьдагийн популяцад харьцангуй бага (9.00) байв. Эффектив аллелийн тоо Алтай (6.73), Говь (8.02), Хорьдол Сарьдагийн (4.2) популяцад хэт бага, Хангайн (9.94) популяцад харьцангуй жигд, хүлээгдэж буй гетерозигот байдал Алтай (0.80, 0.84), Говь (0.74, 0.87), Хангайн (0.85, 0.89) популяцад хамгийн өндөр байв. BM302, BM4505, INRA040 локусуудын генетик олон янз байдал өндөр ч ихэнх аллелиуд нь бага давтамжтай байв. Үүнээс дүгнэхэд, энэхүү боловсруулсан мултиплекс ПГУ амжилттай тогтворжсон бөгөөд олшруулсан бүтээгдэхүүнд фрагмент анализ хийн генетик үзүүлэлтүүдийг тооцож богино хугацаанд популяцийн генетикийн судалгаанд ач холбогдолтой урьдчилсан дүнг гарган авав. Цаашид мултиплекс ПГУ, фрагмент анализийн энэ арга зүйг ашиглан микросателлитын маркеруудын тоог нэмэгдүүлэн судалгааг үргэлжлүүлж аргалийн хамгааллын генетикийн илүү үнэн зөв дүгнэлт гаргах боломжтой гэж үзэв. Түлхүүр үгc: мултиплекс ПГУ тогтворжуулалт, популяцын генетик үзүүлэлт, микросателлит BM302, BM4505, INRA040 локус, аргаль хонь
mutations are shown to be a cause of autism spectrum disorders (ASD) as well as neurodevelopmental disorders (NDD). In conclusion, our results indicate that the hitherto uncharacterized protein FAM124B might be very important for embryonic development and could be involved in the pathogenesis of CHARGE syndrome and ASD/NDDs. Moreover, to analyse the possible role of FAM124B in the pathogenesis of CHARGE and neurocristopathies, we plan to perform a knockdown of FAM124B in Xenopus laevis and test its influence on genes related to neural crest formation. Generation of a knockout mouse model for Fam124B and further characterization of the CHD7 interacting complex members will help to learn more about the molecular mechanism behind CHARGE syndrome and ASD/NDDs. DNA. This mechanism might depend on other proteins, such as histone chaperones or DNA-binding factors, in addition of remodeling proteins, d: creating a loop on the surface of the nucleosome (Adapted and modified from Allis et al. Epigenetics 2007).CHD9 It might be involved in differentiation of osteogenic cells (Shur and Benayahu. 2005, Shur et al. 2006) the authors suggest an important role for CHD7 in gene expression programs for neural crest cell migration and specification (Bajpai et al. 2010). However, a recent study of Randall et al.(2009) demonstrated that a Chd7 rescue in only neural crest cells cannot correct the phenotype of pharyngeal arch defects, while a Chd7 rescue in both neural crest cells and in ectoderm could do it. These findings demonstrate the necessity of CHD7 expression in ectoderm. Role of CHDs in human diseasesTo date, CHD3, CHD4, CHD5, CHD7 and CHD8 are known to be involved in human diseases (Table 2.2). In dermatomyositis, which is a connective tissue disease, CHD3 and CHD4 have been characterized as autoantigens in inflammation of both muscle and skin (Airio et al. 1995, Ge et al. 1995, Seelig et al. 1995, 1996. CHD3 is also suggested to play a role in the pathogenesis of Hodgkin's lymphoma by interacting with Ki-1/57 (Lemos et al. 2003), an intracellular phosphoprotein which is a marker for malignant cells in Hodgkin's lymphoma (Schwab et al. 1982, Rhode et al. 1992.CHD5 is found to be deleted in neuroblastoma and glioma cell lines (Thompson et al. 2003, Law et al. 2005, White et al. 2005. Neuroblastoma is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts. It frequently affects infants, usually under two years old (www.nhs.uk/conditions/Neuroblastoma/Pages/Introduction.aspx). CHD5 is located on chromosome 1p36.31. Deletions of 1p36 are common in human neuronal,
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