Analysis of ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2 in human neural tube defects suggests a possible association with alleles in ALDH1A2

BACKGROUND: Vitamin A (retinol), in the form of retinoic acid (RA), is essential for normal development of the human embryo. Studies in the mouse and zebrafish have shown that retinol is metabolized in the developing spinal cord and must be maintained in a precise balance along the anteroposterior axis. Both excess and deficiency of RA can affect morphogenesis, including failures of neural tube closure. METHODS: We chose to investigate 5 genes involved in the metabolism or synthesis of RA, ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2, for their role in the development of human neural tube defects, such as spina bifida. RESULTS: An association analysis using both allelic and genotypic single-locus tests revealed a significant association between the risk for spina bifida and 3 polymorphisms in the gene ALDH1A2; however, we found no evidence of a significant multilocus association. CONCLUSIONS: These results may suggest that polymorphisms in ALDH1A2 may influence the risk for lumbosacral myelomeningocele in humans.     

Developing with lethal RA levels: genetic ablation of Rarg can restore the viability of mice lacking Cyp26a1

We have previously reported that the retinoic acid (RA) catabolizing enzyme CYP26A1 plays an important role in protecting tail bud tissues from inappropriate exposure to RA generated in the adjacent trunk tissues by RALDH2, and that Cyp26a1-null animals exhibit spina bifida and caudal agenesis. We now show that, in the absence of Cyp26a1, retinoic acid receptor gamma (RARgamma) mediates ectopic RA-signaling in the tail bud. We also show that activated RARgamma results in downregulation of Wnt3a and Fgf8, which integrate highly conserved signaling pathways known for their role in specifying caudal morphogenesis. Ablation of the gene for RARgamma (Rarg) rescues Cyp26a1-null mutant animals from caudal regression and embryonic lethality, thus demonstrating that CYP26A1 suppresses the RA-mediated downregulation of WNT3A and FGF8 signaling pathways by eliminating ectopic RA in gastrulating tail bud mesoderm.     

维甲酸诱导的脊柱裂小鼠的脊髓全基因组表达谱分析(英文)

关于维甲酸胚胎病理学的研究很多,维甲酸受体在器官发生、发育及神经管闭合过程中发挥重要作用。但维甲酸影响这些过程的机制还不清楚。在本研究中,我们发现,小鼠怀孕8天时,给予母体连续3次维甲酸灌胃,将导致胎儿脊柱裂,发生率为96.77%。本研究应用微阵列技术,在维甲酸诱导的脊柱裂小鼠胎儿的脊髓组织中发现了134个差异表达在1.5倍以上的基因。基因富集分析显示,母亲暴露于维甲酸导致的胎儿脊柱裂,与促凋亡和抗凋亡、细胞增殖、迁徙、细胞骨架成分以及细胞或局部粘附等基因功能簇相关,提示这些细胞成分和生物学的功能缺陷促使脊柱发育异常。我们的研究提供了脊柱裂的全基因组基因表达模式,有助于理解神经管缺陷的病因和病理学。     

Genes encoding catalytic subunits of protein kinase A and risk of spina bifida

BACKGROUND: PRKACA and PRKACB are genes encoding the cAMP-dependent protein kinase A (PKA) catalytic subunits alpha and beta, respectively. PKA is known to be involved in embryonic development, as it down-regulates the Hedgehog (Hh) signaling pathway, which is critical to normal pattern formation and morphogenesis. The PKA-deficient mouse model, which has only a single catalytic subunit, provided intriguing evidence demonstrating a relationship between decreased PKA activity and risk for posterior neural tube defects (NTDs) in the thoracic to sacral regions of gene-knockout mice. Unlike most other mutant mouse models of NTDs, the PKA-deficient mice develop spina bifida with 100% penetrance. We hypothesized that sequence variations in human genes encoding the catalytic subunits may alter the PKA activity and similarly increase the risk of spina bifida. METHODS: We sequenced the coding regions and the exon/intron boundaries of PRKACA and PRKACB. We also examined 3 common single-nucleotide polymorphisms (SNPs) of these 2 genes by allele discrimination. RESULTS: Five sequence variants in coding region and 2 intronic sequence variants proximal to exons were detected. None of the 3 SNPs examined in the association study appeared to be associated with substantially increased risk for spina bifida. CONCLUSIONS: Our results did not reveal a strong association between these PKA SNPs and spina bifida risk. Nonetheless, it is important to examine the possible gene-gene interactions between PRKACA and PRKACB when evaluating the risk for NTDs, as well as genes encoding regulatory subunits of PKA. In addition, interactions with other genes such as Sonic Hedgehog (SHH) should also be considered for future investigations.     

Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome

BACKGROUND: The retinoic acid (RA)-catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1-null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS: Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by chi(2) analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS: Mutational screening allowed us to identify 6 SNPs, 4 of which (447 C>G, 1134 G>A, IVS 1+10 G>C, and IVS 4+8 AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS: Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects.     

Interaction of PDGFRA promoter haplotypes and maternal environmental exposures in the risk of spina bifida

BACKGROUND: Neural tube defects are multifactorial malformations involving both environmental exposures, such as maternal nutrition, and genetic factors. Aberrant expression of the platelet‐derived growth factor alpha‐receptor (PDGFRA) gene has been implicated in neural‐tube‐defect etiology in both mice and humans. METHODS: We investigated possible interactions between the PDGFRA promoter haplotype of mother and child, as well as maternal glucose, myo‐inositol, and zinc levels, in relation to spina bifida offspring. Distributions were determined of the PDGFRA promoter haplotypes H1 and H2 in a Dutch cohort, consisting of 88 spina bifida children with 56 of their mothers, and 74 control children with 72 of their mothers, as well as maternal plasma glucose, myo‐inositol, and red blood cell zinc concentrations. RESULTS: A significantly higher frequency of H1 was observed in children with spina bifida than in controls (30.1 vs. 20.3%; OR = 1.69, 95% CI 1.02–2.83). High maternal body mass index (BMI) and glucose were significant risk factors for both H1 and H2 children, whereas low myo‐inositol and zinc were risk factors for H2 but not for H1 children. Stepwise multiple logistic regression analysis showed that high maternal glucose and low myo‐inositol are the main risk factors for H2 spina bifida children, whereas for H1 spina bifida children, maternal BMI was the main risk factor. Interestingly, H1 mothers (median 165.5 cm) showed a significantly lower body height than H2 mothers (median 169.1 cm; p = 0.003). CONCLUSIONS: These data suggest that the child's PDGFRA promoter haplotype is differentially sensitive for periconceptional exposure to glucose, myo‐inositol, and zinc in the risk of spina bifida. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Spina bifida aperta induced by valproic acid and by all-trans-retinoic acid in the mouse: distinct differences in morphology and periods of sensitivity.

The antiepileptic drug valproic acid (VPA) has been implicated as a human teratogen causing spina bifida aperta. Recently, we developed a mouse model inducing spina bifida aperta with VPA. To elucidate the pathogenesis of VPA-induced spina bifida aperta we now investigated the anatomy and histology of this defect in the mouse. The morphology of spina bifida aperta induced by all-trans-retinoic acid (RA) was used for comparison. Various doses of VPA and RA were administered at different times to determine the periods of sensitivity for inducing spina bifida aperta with these drugs. Each administration regimen consisted of three doses applied at intervals of 6 hr. RA induced spina bifida aperta during an earlier developmental period (day 8 of gestation) than VPA (day 9 of gestation). The most effective regimens for induction of spina bifida aperta in mice were injections of 3 x 500 mg VPA-Na/kg body weight (b.w.) intraperitoneally on day 9 of gestation at 0, 6, and 12 hr; RA (12.5 mg/kg b.w.) was given orally on day 8 of gestation at 12 and 18 hr, day 9 at 0 hr. VPA did not induce spina bifida aperta on day 8 of gestation and RA did not induce this effect on day 9 of gestation. Histological studies of day 18 fetuses carrying spina bifida aperta were performed. The spina bifida aperta induced by VPA shows a disorganized and necrotic spinal cord. In the vertebral canal were observed cell debris, blood cells, capillaries, macrophages, and rests of meninges. These results indicate that the spinal cord is almost destroyed at the affected section. In contrast, the spina bifida aperta induced by RA demonstrates a spinal cord organized in the gray and white matter, the dorsal and ventral horn. But the neural canal does not exist, only a layer of ependymal cells lies on the surface of the spinal cord. Our results indicate that the morphology of spina bifida aperta induced by VPA differed distinctly from that induced by RA in the mouse fetus. Moreover VPA produced a spina bifida aperta with a specific morphology. Also the period of sensitivity for induction of this lesion differed and occurred earlier for RA than for VPA. VPA and RA may possibly induce spina bifida aperta via different mechanisms in the mouse.     

Quantification and localization of expression of the retinoic acid receptor-beta and -gamma mRNA isoforms during neurulation in mouse embryos with or without spina bifida

BACKGROUND: Previous studies observed that retinoic acid receptor-gamma (RARgamma) is expressed in the open caudal neuroepithelium but that RARbeta is expressed in the closed neural tube. Furthermore, retinoic acid (RA) induces RARbeta expression, a molecular event associated with neural tube closure, but treatment with RA at the appropriate gestation time causes failure of neural tube closure. Since there are four isoforms of RARbeta, perhaps the isoforms expressed in the closed neural tube and induced by RA are different. To investigate the hypothesis that the switch from RARgamma to RARbeta is mechanistically linked to neural tube closure, this study determined the concentrations and distributions of RARbeta and RARgamma isoforms in mouse embryos with RA-induced neural tube defects and in splotch (Sp) mutant embryos with spina bifida. METHODS: Absolute concentrations of RARbeta and RARgamma isoforms were determined throughout primary neurulation (gestational day 8.5-10.0) in treated or untreated C57BL/6J mouse whole embryos by ribonuclease protection analysis. Treatment consisted of an oral dose of 100 mg/kg of all-trans-RA on gestational day 8.5. Spatial distributions of RARbeta and RARgamma were examined in RA-treated and Sp mutant embryos by in situ hybridization. RESULTS: RARbeta2, gamma1, and gamma2 were expressed in untreated embryos and were induced 4.5-, 1.6-, and 4.0-fold, respectively, 4 hr after treatment with RA. In embryos with RA-induced spina bifida, RARbeta2 was expressed in the closed neural tube while RARgamma1 and RARgamma2 were expressed in the open caudal neuroepithelium. In splotch mice with spina bifida, the boundary between RARbeta and RARgamma did not correspond to the site of neural tube closure. CONCLUSIONS: In RA-treated embryos, the relationship between RARbeta expression in the closed and RARgamma in the open caudal neuroepithelium was not altered. However, in splotch embryos with spina bifida, the juncture between RARbeta and RARgamma expression remained in the same anatomical position in the neuroepithelium irrespective of the neural tube closure status and suggests that the switch from RARgamma to RARbeta expression in the closing caudal neuroepithelium may not be causally linked to neural tube closure in the splotch mutant.     

Proteomic analysis of amniotic fluid of pregnant rats with spina bifida aperta

Congenital spina bifida aperta is a common congenital malformation in children and has an incidence of 1‰ to 5‰ in China. However, we currently lack specific biomarkers for screening or prenatal diagnosis and there is no method to entirely cure or prevent such defects. In this study, we used two-dimensional gel electrophoresis (2-DE)/mass spectrometry (MS) to characterize differentially expressed proteins in amniotic-fluid samples (AFSs) of embryonic day (E) 17.5 rat fetuses with spina bifida aperta induced by retinoic acid (RA). We identified five proteins differentially expressed in AFSs of spina bifida aperta, including three upregulated proteins (transferrin, alpha-1 antiproteinase and signal recognition particle receptor, B subunit [SRPRB] 55 kDa), two downregulated proteins (apolipoprotein A IV [APO A4] and Srprb 77 kDa). Specifically, we found 11 alpha-1 fetoprotein (AFP) fragments that were downregulated and 35 AFP fragments that were upregulated in AFSs from embryos with spina bifida aperta. Of the downregulated AFP fragments, 72.7% (8/11) were confined to the AFP N-terminus (amino acids [aas] 25-440) and 77.1% (27/35) of upregulated AFP fragments were confined to the AFP C-terminus (aas 340-596). We also confirmed APO A4 and AFP by immunoblot analysis. This is the first comparative proteomic study of AFSs from rat fetuses with spina bifida aperta. We demonstrate proteomic alterations in the AFS of spina bifida aperta, which may provide new insights in neural tube defects and contribute to the prenatal screening.     

Retinoids, eye development, and maturation of visual function

Vitamin A is known to be critical for the beginning of eye development as well as for photoreception in the functional retina. Hardly anything, however, is known about whether retinoic acid (RA)-regulated gene expression also plays a role in the long intervening period, during which the neurobiological retinal structure takes shape. The eye contains a highly intricate architecture of RA-synthesizing (RALDH) and degrading (CYP26) enzymes. Whereas the RALDHs are integrated in the early molecular mechanisms through which the dorso-ventral retina organization is established, the CYP26 enzymes are not necessary for this process and no molecular targets that match their retinal expression pattern have yet been identified. In this article we describe that CYP26 expression in the mouse is most distinctive during later stages of retina formation. Throughout development CYP26A1 degrades RA in a horizontal region that extends across the retina, but during later embryonic and postnatal retina maturation this function is reinforced by another enzyme, CYP26C1. RA applications at this stage do not affect the RALDHs but cause differential changes in CYP26 expression: Cyp26a1 is up-regulated, but more rapidly by 9-cis than all-trans RA, Cyp26c1 is down-regulated, and Cyp26b1, which is undetectable in the normal mouse retina, is strongly activated in retinal ganglion cells. The dynamic regulation in RA-difference patterns by the CYP26 enzymes may set up spatial constellations for expression of genes involved in formation of retinal specializations for higher acuity vision, which are known to form over a prolonged period late in retina development.     

The involvement of cytochrome p450 (CYP) 26 in the retinoic acid metabolism of human epidermal keratinocytes

All-trans retinoic acid (RA) levels are controlled by enzymes of the vitamin A metabolism (RDH16, RalDH2, and LRAT) and RA catabolism (CYP26 and CYP2S1). Here, the mRNA expression of these enzymes was investigated in human keratinocytes at different Ca²⁺concentrations and after exposure to RA and CYP26 inhibitors. Cellular differentiation (high Ca²⁺) increased the expression of LRAT, RDH16 and RalDH2, and decreased CYP26B1. RA (1 μM) induced CYP26A1, CYP26B1, CYP2S1, CRABPII and LRAT mRNA. The CYP26 inhibitor talarozole altered CYP26A1 and LRAT mRNA expression in a similar way as RA, increased the cellular accumulation of [³H]RA, and induced a punctate CRABPII staining, also observed after siRNA knock-down of CYP26B1 (but not after RA exposure). Furthermore, CYP26B1 siRNA increased the accumulation of [³H]RA and the CRABPII mRNA, suggesting an augmented retinoid signalling. Thus CYP26B1 appears essential for RA catabolism under physiological conditions, whereas CYP26A1 might play a greater role during RA excess.     

Analysis of Cyp26b1/Rarg compound-null mice reveals two genetically separable effects of retinoic acid on limb outgrowth

The role of retinoic acid (RA) in limb development is unclear, although it has been suggested to be a proximalizing factor which plays a morphogenetic role in pattern formation. Exogenous RA produces a teratogenic effect on limb morphology; similarly, changes in the endogenous distribution of RA following genetic ablation of the RA-metabolizing enzyme, CYP26B1, result in phocomelia accompanied by changes in expression of proximo-distal (P-D) patterning genes, increased cell death, and delayed chondrocyte maturation. Here we show that disruption of RA receptor (RAR) gamma in a Cyp26b1^−/− background is able to partially rescue limb skeletal morphology without restoring normal expression of proximo-distal patterning genes. We further show that embryos deficient in CYP26B1 exhibit early localized domains of mesenchymal cell death, which are reduced in compound-null animals. This model reveals two genetically separable effects of RA in the limb: an apoptotic effect mediated by RARγ in the presence of ectopic RA, and a P-D patterning defect which is uncovered following the loss of both CYP26B1 and RARγ. These data provide genetic evidence to clarify the roles of both RA and CYP26B1 in limb outgrowth and proximo-distal patterning.     

微卫星不稳定性与脊柱裂发生的关联性研究

目的：通过对脊柱裂（spina bifida）胚胎脑组织中微卫星不稳定性（microsatellite instability,MSI）的分析,探讨遗传不稳定性是否为此疾病的特征之一,进而研究错配修复系统（mismatch repair,MMR）与脊柱裂发病的分子机制。方法：19例脊柱裂和19例非神经管畸形对照脑组织中提取DNA;尿素变性凝胶电泳法检测标本中MSI发生情况。结果：在19例脊柱裂脑组织中9例MSI阳性,阳性率47.4%。其中2例为高度微卫星不稳定（high frequency microsatellite instability,MSI-H）,7例为低度微卫星不稳定（lowfrequency microsatellite instability,MSI-L）,其余10例为微卫星稳定（microsatellite stable,MSS）,对照组中未出现MSI。选择的5个微卫星位点MSI的阳性率分别为Bat34C4（10.5%）、Bat26（26.5%）、D2S123（10.5%）、D3S1611（5.3%）,D2S119（5.3%）。结论：脊柱裂中存在MSI现象,提示MSI、错配修复系统可能与脊柱裂的发生有一定关系。     

Cloning of rat cytochrome P450RAI (CYP26) cDNA and regulation of its gene expression by all-trans-retinoic acid in vivo

A novel retinoic acid (RA)-inducible cytochrome P450 (P450 RAI or CYP26), previously cloned from human, zebra fish, and mouse, functions in the metabolism of all-trans-RA to polar metabolites including 4-hydroxy-RA and 4-oxo-RA. To further study CYP26 in the rat model, we first cloned rat CYP26 cDNA. The nucleotide sequence predicts a 497-amino-acid protein whose sequence is 95% identical to mouse and 91% homologous to human CYP26. Animal studies showed that CYP26 mRNA expression is very low (0.01+/-0.008;P<0.05) in vitamin-A-deficient rats compared to pair-fed vitamin-A-sufficient rats (defined as 1.0). In a kinetic study, vitamin-A-deficient rats were treated with approximately 100 microg of all-trans-RA and liver was collected after 3-72 h for analysis of CYP26 mRNA by quantitative real-time PCR. Liver CYP26 mRNA increased to nearly 10-fold above control after 3 h (P<0.01), reaching a peak of about 2000-fold greater around 10 h (P<0.001) and then decreased rapidly. The CYP26 dose response to RA was nearly linear (R(2)=0.9638). Additionally, significant regulation of CYP26 gene expression was observed in the vitamin-A-deficient, control, and RA-treated condition in lung, testis, and small intestine. We conclude that CYP26 mRNA expression is dynamically regulated in vivo by diet and RA in hepatic and extrahepatic tissues. The long-term down-regulation of CYP26 in retinoid deficiency may be critical for conserving RA, while the acute up-regulation of CYP26 may be important for preventing a deleterious overshoot of RA derived from either dietary or exogenous sources.     

Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.     

Genetic polymorphisms and haplotype structures of the CYP4A22 gene in a Japanese population

The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.     

In a shake of a lamb's tail: using genomics to unravel a cause of chondrodysplasia in Texel sheep

Chondrodysplasia in Texel sheep is a recessively inherited disorder characterized by dwarfism and angular deformities of the forelimbs. A genome-wide association study using the Illumina OvineSNP50 BeadChip on 15 sheep diagnosed as affected and eight carriers descended from three affected rams was conducted to uncover the genetic cause. A homozygous region of 25 consecutive single nucleotide polymorphism (SNP) loci was identified in all affected sheep, covering a region of 1 Mbp on ovine chromosome 4. Seven positional candidate genes - including the solute carrier family 13 (sodium/sulphate symporters), member 1 (SLC13A1) - were identified and used to search for new SNPs for fine mapping of the causal locus. The SLC13A1 gene, encoding a sodium/sulphate transporter, was the primary candidate gene attributable to similar phenotypes observed in the Slc13a1 knockout mouse model. We discovered a 1-bp deletion of T (g.25513delT) at the 107 bp position of exon 3 in the SLC13A1 gene. Genotyping by direct sequencing and restriction fragment length polymorphism analysis for this mutation showed that all 15 affected sheep were g.25513delT/g.25513delT; the eight carriers were g.25513delT/T and 54 normal controls were T/T. The mutation g.25513delT shifts the open reading frame of SLC13A1 to introduce a stop codon and truncate C-terminal amino acids. It was concluded that the g.25513delT mutation in the SLC13A1 gene was responsible for the chondrodysplasia seen in these Texel sheep. This knowledge can be used to identify carriers with the defective g.[25513delT] allele to avoid at-risk matings to improve animal welfare and decrease economic losses.