Genetic research progress in branchio-oto syndrome/ branchio-oto-renal syndrome. / 鳃耳综合征/é³ƒè€³è‚¾ç»¼åˆå¾çš„é—ä¼ å­¦ç ”ç©¶è¿›å±•.

Branchio-oto syndrome (BOS)/branchio-oto-renal syndrome (BORS) is a kind of autosomal dominant heterogeneous disorder. These diseases are mainly characterized by hearing impairment and abnormal phenotype of ears, accompanied by renal malformation and branchial cleft anomalies including cyst or fistula, with an incidence of 1/40 000 in human population. Otic anormalies are one of the most obvious clinical manifestations of BOS/BORS, including deformities of external, middle, inner ears and hearing loss with conductive, sensorineural or mix, ranging from mild to profound loss. Temporal bone imaging could assist in the diagnosis of middle ear and inner ear malformations for clinicians. Multiple methods including direct sequencing combined with next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), or array-based comparative genomic hybridization (aCGH) can effectively screen and identify pathogenic genes and/or variation types of BOS/BORS. About 40% of patients with BOS/BORS carry aberrations of EYA1 gene which is the most important cause of BOS/BORS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements. Human Endogenous Retroviral sequences (HERVs) may play an important role in mediating EYA1 chromosomal fragment deletion mutations caused by non-allelic homologous recombination. EYA1 encodes a phosphatase-transactivator cooperated with transcription factors of SIX1, participates in cranial sensory neurogenesis and development of branchial arch-derived organs, then regulates the morphological and functional differentiation of the outer ear, middle ear and inner ear toward normal tissues. In addition, pathogenic mutations of SIX1 and SIX5 genes can also cause BOS/BORS. Variations of these genes mentioned above may cause disease by destroying the bindings between SIX1-EYA1, SIX5-EYA1 or SIX1-DNA. However, the role of SIX5 gene in the pathogenesis of BORS needs further verification.

Exome-wide Association Study Identifies GREB1L Mutations in Congenital Kidney Malformations.

Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10-5 for novel LOF, increased to p = 4.1 × 10-6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10-7). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10-12). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.

Study of expression of genes potentially responsible for reduced fitness in patients with myotonic dystrophy type 1 and identification of new biomarkers of testicular function.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by trinucleotide CTG expansion in DMPK gene, often affecting the neighboring genes. Endocrine system is involved, resulting in hypogonadism and reproductive abnormalities, but molecular mechanisms underlying the reduced fertility observed in DM1 are very complex and partially unknown. To better characterize these mechanisms, an analysis of sperm parameters and anti-Müllerian hormone (AMH) values was performed in 20 DM1 patients. About 50% of them showed hypoposia and azoospermia; the remaining, despite an adequate volume of ejaculate, had oligo-astheno-teratozoospermia. Interestingly, the lowest AMH levels better correlated with the main sperm alterations. The pattern of expression of DMPK, SIX5, and RSPH6A genes, evaluated by quantitative reverse transcription polymerase chain reaction, showed a substantial reduction of the expression in both peripheral blood and in seminal plasma of patients, compared to controls. An impairment of testis-specific RSPH6A protein expression and localization was observed in sperm protein extracts by WB analysis and in isolated spermatozoa by immunofluorescence. These results support the hypothesis that CTG expansion also affects the expression of neighboring genes and contributes to gonad defects observed in DM1, suggesting the possibility of using them as markers for normal fertility in humans.

Low Six4 and Six5 gene dosage improves dystrophic phenotype and prolongs life span of mdx mice.

Muscle regeneration is an important process for skeletal muscle growth and recovery. Repair of muscle damage is exquisitely programmed by cellular mechanisms inherent in myogenic stem cells, also known as muscle satellite cells. We demonstrated previously the involvement of homeobox transcription factors, SIX1, SIX4 and SIX5, in the coordinated proliferation and differentiation of isolated satellite cells in vitro. However, their roles in adult muscle regeneration in vivo remain elusive. To investigate SIX4 and SIX5 functions during muscle regeneration, we introduced knockout alleles of Six4 and Six5 into an animal model of Duchenne Muscular Dystrophy (DMD), mdx (Dmd(mdx) /Y) mice, characterized by frequent degeneration-regeneration cycles in muscles. A lower number of small myofibers, higher number of thick ones and lower serum creatine kinase and lactate dehydrogenase activities were noted in 50-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice than Dmd(mdx) /Y mice, indicating improvement of dystrophic phenotypes of Dmd(mdx) /Y mice. Higher proportions of cells positive for MYOD1 and MYOG (markers of regenerating myonuclei) and SIX1 (a marker of regenerating myoblasts and newly regenerated myofibers) in 12-week-old Six4(+/-) 5(+/-) Dmd(mdx) /Y mice suggested enhanced regeneration, compared with Dmd(mdx) /Y mice. Although grip strength was comparable in Six4(+/-) 5(+/-) Dmd(mdx) /Y and Dmd(mdx) /Y mice, treadmill exercise did not induce muscle weakness in Six4(+/-) 5(+/-) Dmd(mdx) /Y mice, suggesting higher regeneration capacity. In addition, Six4(+/-) 5(+/-) Dmd(mdx) /Y mice showed 33.8% extension of life span. The results indicated that low Six4 and Six5 gene dosage improved dystrophic phenotypes of Dmd(mdx) /Y mice by enhancing muscle regeneration, and suggested that SIX4 and SIX5 are potentially useful de novo targets in therapeutic applications against muscle disorders, including DMD.

Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.

UNLABELLED: Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.

The SIX5 Protein in Fusarium oxysporum f. sp. cepae Acts as an Avirulence Effector toward Shallot (Allium cepa L. Aggregatum Group).

Fusarium oxysporum f. sp. cepae (Foc) causes basal rot disease in Allium species, including onions (Allium cepa L.) and shallots (A. cepa L. Aggregatum group). Among Allium species, shallots can be crossbred with onions and are relatively more resistant to Foc than onions. Thus, shallots are considered a potential disease-resistant resource for onions. However, the mechanisms underlying the molecular interactions between shallots and Foc remain unclear. This study demonstrated that SIX5, an effector derived from Foc (FocSIX5), acts as an avirulence effector in shallots. We achieved this by generating a FocSIX5 gene knockout mutant in Foc, for which experiments which revealed that it caused more severe wilt symptoms in Foc-resistant shallots than the wild-type Foc and FocSIX5 gene complementation mutants. Moreover, we demonstrated that a single amino acid substitution (R67K) in FocSIX5 was insufficient to overcome shallot resistance to Foc.

Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression.

Background: The transcription partners of eyes absent homologs and sine oculis homeobox homologs (EYA-SIX) contribute to tumorigenesis and progression of multiple cancers through mediating the expression of oncogenes and tumor suppressors. This study aimed to determine the roles of individual EYA-SIX partners and their downstream targets in colorectal cancer (CRC). Methods: Immunoblot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to measure protein and gene expression levels. Cell Counting Kit-8 (CCK-8) assay, colony formation, cell invasion assays, and a tumor xenograft model were chosen to investigate tumor cell growth. Immunoprecipitation, mass spectrometry, and co-immunoprecipitation (Co-IP) experiments were performed to determine the assembly of the SIX5-associated complex. Chromatin immunoprecipitation (ChIP) assay was used to evaluate the occupancy of SIX5-associated complex on its target gene promoters. Results: We discovered that the hypoxia-induced EYA3 coupled with SIX5 and a histone acetyltransferase p300 to assemble a complex in CRC biopsies. The EYA3-SIX5-p300 complex was required for the transactivation of epidermal growth factor receptor (EGFR), vascular endothelial growth factor D (VEGFD), and five matrix metallopeptidases (MMPs), including MMP3, MMP7, MMP8, MMP21, and MMP26. The results of ChIP revealed that the EYA3-SIX5-p300 complex specifically bound to the promoters of EGFR/VEGFD/MMPs. Disruption of the assembly of EYA3-SIX5-p300 complex decreased the expression of EGFR/VEGFD/MMPs, inhibiting CRC cell growth. Administration of EYA3 inhibitor (benzarone) in mice harboring tumor xenografts significantly inhibited tumor growth. Conclusions: The hypoxia-dependent EYA3-SIX5-p300 complex is involved in the pathogenesis of CRC through mediating EGFR/VEGFD/MMPs and targeting this complex may represent a new therapeutic strategy for CRC treatment.

Mice doubly deficient in Six4 and Six5 show ventral body wall defects reproducing human omphalocele.

Omphalocele is a human congenital anomaly in ventral body wall closure and may be caused by impaired formation of the primary abdominal wall (PAW) and/or defects in abdominal muscle development. Here, we report that mice doubly deficient in homeobox genes Six4 and Six5 showed the same ventral body wall closure defects as those seen in human omphalocele. SIX4 and SIX5 were localized in surface ectodermal cells and somatic mesoderm-derived mesenchymal and coelomic epithelial cells (CECs) in the PAW. Six4-/-;Six5-/- fetuses exhibited a large omphalocele with protrusion of both the liver and intestine, or a small omphalocele with protrusion of the intestine, with complete penetrance. The umbilical ring of Six4-/-;Six5-/- embryos was shifted anteriorly and its lateral size was larger than that of normal embryos at the E11.5 stage, before the onset of myoblast migration into the PAW. The proliferation rates of surface ectodermal cells in the left and right PAW and somatic mesoderm-derived cells in the right PAW were lower in Six4-/-;Six5-/- embryos than those of wild-type embryos at E10.5. The transition from CECs of the PAW to rounded mesothelial progenitor cells was impaired and the inner coelomic surface of the PAW was relatively smooth in Six4-/-;Six5-/- embryos at E11.25. Furthermore, Six4 overexpression in CECs of the PAW promoted ingression of CECs. Taken together, our results suggest that Six4 and Six5 are required for growth and morphological change of the PAW, and the impairment of these processes is linked to the abnormal positioning and expansion of the umbilical ring, which results in omphalocele.