A frameshift mutation in the HuP2 paired domain of the probable human homolog of murine Pax-3 is responsible for Waardenburg syndrome type 1 in an Indonesian family.

Waardenburg syndrome type 1 (WS1) is an autosomal dominant disorder characterized by deafness, dystopia canthorum, heterochromia iridis, white forelock, and premature greying. A similar phenotype is caused in the mouse by mutations in the Pax-3 gene. This observation, together with comparisons of conserved syntenies in the murine and human genetic maps, suggested that at least some WS1 mutations should occur in HuP2, the probable human homolog of Pax-3. Two mutations in the HuP2 sequence of individuals with WS1 have been reported recently. Both of them occur in the highly conserved paired box region of the gene, which encodes a DNA binding domain. The functional consequences of these mutations are at present speculative. We report here a 14 bp deletion in the paired domain encoded by exon 2 of HuP2 in an Indonesian family segregating for WS1. This frameshift mutation results in a premature termination codon in exon 3. The HuP2 product is a truncated protein lacking most of the paired domain and all of the predicted homeo domain. We propose that the WS1 phenotype in this family is due to loss of function of HuP2 and discuss two mechanisms for the dominant effect of this mutation.     

The paired box encodes a second DNA-binding domain in the paired homeo domain protein.

The homeo box, which encodes the DNA-binding homeo domain, is a DNA sequence motif present in several Drosophila developmental genes; it has been used to identify many homologous genes involved in mammalian development. The paired box is another conserved sequence motif, first identified in the paired (prd) and gooseberry (gsb) Drosophila homeo domain genes. It encodes a 128-amino-acid domain, the paired domain, which has since been found in other fly and mouse gene products, in association with the homeo domain or in its absence. We show that the paired box of the prd gene encodes a DNA-binding activity, independent of the DNA-binding activity of the Paired (Prd) homeo domain and with a different sequence specificity. The amino-terminal region of the paired domain, including one of the three predicted alpha-helices, is necessary and sufficient for binding. We investigate the binding of the Prd protein to two sites in the even-skipped promoter, which are composed of overlapping sequences bound by the homeo domain and by the paired domain. We also show that a mutation in the paired box of Prd, corresponding to the mutation in the paired box of the mouse Pax-1 gene thought to cause the undulated skeletal phenotype, destroys the ability of the Prd protein to bind to the paired domain-specific site. This supports the view that the undulated phenotype results from the inactivation of the DNA-binding activity of the paired domain of Pax-1.     

Biochemical defects in eight SRY missense mutations causing XY gonadal dysgenesis

Sex determining Region of the Y chromosome (SRY) is the Y-borne gene required for male sex determination. Many XY females with complete gonadal dysgenesis carry SRY mutations. We describe here the effects of eight clinically isolated point mutations on the DNA-binding and -bending functions of SRY. We found that the seven mutations in the HMG domain affected the protein's DNA-binding and -bending activities to varying degrees, although all cause complete gonadal dysgenesis. DNA binding was abolished by the R75N and L94P mutations, severely disrupted by the F67V mutation and reduced by the M64R (6-fold), R76P (4-fold), A113T (3-fold), and M78T (1.7-fold) mutations. Of these, variant M64R showed no DNA-bending activity, while M78T caused a mild reduction in DNA bending. The S18N mutation, a familial mutation that lies outside the HMG domain and caused partial gonadal dysgenesis in one patient, had minimal effect on DNA binding and bending. Analysis of the NMR solution structure of the SRY HMG domain bound to DNA suggests that mutations disrupt the protein's conformation (helicity, packing), or interactions at the DNA interface. The degree to which mutations causing complete gonadal dysgenesis affect the DNA-binding activity varies. We propose that there is a threshold level of SRY activity or expression required for testis determination, as we observe that familial mutations have the least effect on SRY activity.     

VSX1: a gene for posterior polymorphous dystrophy and keratoconus

We identified mutations in the VSX1 homeobox gene for two distinct inherited corneal dystrophies; posterior polymorphous dystrophy (PPD) and keratoconus. One of the mutation (R166W) responsible for keratoconus altered the homeodomain and impaired DNA binding. Two other sequence changes (L159M and G160D) were associated with keratoconus and PPD, respectively, and involved a region adjacent to the homeodomain. The G160D substitution, and a fourth defect affecting the highly conserved CVC domain (P247R), occurred in a child with very severe PPD who required a corneal transplant at 3 months of age. In this family, relatives with the G160D change alone had mild to moderate PPD, while P247R alone caused no corneal abnormalities. However, with either the G160D or P247R mutation, electroretinography detected abnormal function of the inner retina, where VSX1 is expressed. These data define the molecular basis of two important corneal dystrophies and reveal the importance of the CVC domain in the human retina.     

Functional analysis of paired box missense mutations in the PAX6 gene

Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, we performed a functional analysis of two missense mutations in the paired domain: the R26G mutation, previously reported in a case of Peters' anomaly, and an unreported I87R mutation, which we identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. We showed that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. Our data support the haploid-insufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele.      

Functional analyses of two newly identified PITX2 mutants reveal a novel molecular mechanism for Axenfeld-Rieger syndrome

The specific role of PITX2 in the pathogenesis of anterior segment dysgenesis has yet to be clearly defined. We provide here new insight into PITX2 pathogenesis through mutational and functional analyses. Three PITX2 mutations were found in a screen of 38 unrelated individuals affected with anterior segment anomalies (8%). All three mutations were found among the 21 individuals affected with Axenfeld-Rieger syndrome (ARS). We have identified two novel mutations, a valine-->leucine (V45L) missense mutation at position 45 within the PITX2 homeodomain, and a seven amino acid duplication (7aaDup) of residues 6-12 of the homeodomain. DNA-binding studies of the two mutant PITX2 proteins demonstrated a <10-fold reduction in the DNA-binding activity of the V45L mutant, and a >100-fold reduction in activity of the 7aaDup mutant. Luciferase reporter assays showed a >200% increase in PITX2 transactivation activity of the V45L mutant, while the 7aaDup mutant was unable to transactivate at detectable levels. Our analyses of the V45L PITX2 mutant reveal that the DNA-binding domain of PITX2 can influence transactivation activity independently of DNA binding. Furthermore, our findings expand the hypothesis that the amount of residual PITX2 activity underlies the variable severity of ocular phenotypes that result from PITX2 mutation. For the first time, we present evidence that increased PITX2 activity may underlie the severe ARS ocular phenotype. We conclude that increased activity of one PITX2 allele may be as physiologically disruptive as a mutation that nullifies a PITX2 allele, with either condition resulting in ARS.     

Molecular analysis of a human PAX6 homeobox mutant

Pax6 controls eye, pancreas and brain morphogenesis. In humans, heterozygous PAX6 mutations cause aniridia and various other congenital eye abnormalities. Most frequent PAX6 missense mutations are located in the paired domain (PD), while very few missense mutations have been identified in the homeodomain (HD). In the present report, we describe a molecular analysis of the human PAX6 R242T missense mutation, which is located in the second helix of the HD. It was identified in a male child with partial aniridia in the left eye, presenting as a pseudo-coloboma. Gel-retardation assays revealed that the mutant HD binds DNA as well as the wild-type HD. In addition, the mutation does not modify the DNA-binding properties of the PD. Cell transfection assays indicated that the steady-state levels of the full length mutant protein are higher than those of the wild-type one. In cotransfection assays a PAX6 responsive promoter is activated to a higher extent by the mutant protein than by the wild-type protein. In vitro limited proteolysis assays indicated that the presence of the mutation reduces the sensitivity to trypsin digestion. Thus, we suggest that the R242T human phenotype could be due to abnormal increase of PAX6 protein, in keeping with the reported sensitivity of the eye phenotype to increased PAX6 dosage.     

婴儿严重肌阵挛癫痫钠离子通道SCNIA基因突变分析

目的 研究婴儿严重肌阵挛癫痫(severe myoclonic epilepsy of infancy,SMEI)患儿钠离子通道a1亚单位基因(sodium channel al subunit gene,SCNIA)突变筛查及遗传特征.方法 收集SMEI患儿23例及其家系成员的临床资料及外周血DNA,采用PCR扩增和DNA直接测序的方法筛查SCNIA基因的26个外显子的突变.结果 23例SMEI患儿中17例有SCNlA基因突变.基因突变率约为73.9%(17/23),其中8例为错义突变(F90S、I91T、A239T、W952G、T1210K,V1335M、V1390M、G1433E),3例为无义突变(R612X、W768X、w1408X),3例为缺失突变(A395fsX400、L556fsX557、V1778fsX1800),1例为插入突变(Y1241fsX1270),1例为剪切部位突变(IVS10+3A>G),1例为同义突变(K1492K),截断突变约占总突变的47.1%(8/17).13个突变位点(F90S、I91T、T1210K、V1335M、G1433E、R612X、W768X,A395faX400、L556fsx557、V1778fsXl800、Y1241fsXl270、IVS10+3A>G、K1492K)经相关检索未见报道.14例突变已证实为新生突变,其余3例突变尚不能确定突变来源.结论 SCNIA基因是SMEI患儿的主要致病基因,约一半为截断突变.SMEI患儿的SCNIA基因突变无热点,且多为新生突变.