Mutations in the human SIX3 gene in holoprosencephaly are loss of function

Holoprosencephaly (HPE) is the most common developmental anomaly of the human forebrain; however, the genetics of this heterogeneous and etiologically complex malformation is incompletely understood. Heterozygous mutations in SIX3, a transcription factor gene expressed in the anterior forebrain and eyes during early vertebrate development, have been frequently detected in human HPE cases. However, only a few mutations have been investigated with limited functional studies that would confirm a role in HPE pathogenesis. Here, we report the development of a set of robust and sensitive assays of human SIX3 function in zebrafish and apply these to the analysis of a total of 46 distinct mutations (19 previously published and 27 novel) located throughout the entire SIX3 gene. We can now confirm that 89% of these putative deleterious mutations are significant loss-of-function alleles. Since disease-associated single point mutations in the Groucho-binding eh1-like motif decreases the function in all assays, we can also confirm that this interaction is essential for human SIX3 co-repressor activity; we infer, in turn, that this function is important in HPE causation. We also unexpectedly detected truncated versions with partial function, yet missing a SIX3-encoded homeodomain. Our data indicate that SIX3 is a frequent target in the pathogenesis of HPE and demonstrate how this can inform the genetic counseling of families

The mutational spectrum of holoprosencephaly-associated changes within the SHH gene in humans predicts loss-of-function through either key structural alterations of the ligand or its altered synthesis.

International audienceMutations within either the SHH gene or its related pathway components are the most common, and best understood, pathogenetic changes observed in holoprosencephaly patients; this fact is consistent with the essential functions of this gene during forebrain development and patterning. Here we summarize the nature and types of deleterious sequence alterations among over one hundred distinct mutations in the SHH gene (64 novel mutations) and compare these to over a dozen mutations in disease-related Hedgehog family members IHH and DHH. This combined structural analysis suggests that dysfunction of Hedgehog signaling in human forebrain development can occur through truncations or major structural changes to the signaling domain, SHH-N, as well as due to defects in the processing of the mature ligand from its pre-pro-precursor or defective post-translation bi-lipid modifications with palmitate and cholesterol

Detection of mutations in GATA1 gene using automated denaturing high-performance liquid chromatography and direct sequencing in children with Down syndrome

Denaturing high-performance liquid chromatography (dHPLC) was developed to screen DNA variations by separating heteroduplex and homoduplex DNA fragments by ion-pair reverse-phase liquid chromatography. in this study, we have evaluated the dHPLC screening method and direct sequencing for the detection of GATA1 mutations in peripheral blood and bone marrow aspirates samples from children with Down syndrome (DS). Cases were ascertained consecutively as part of an epidemiological study of DS and hematological disorders in Brazil. A total of 130 samples corresponding to 115 children with DS were analysed using dHPLC and direct sequencing methods to detect mutations in GATA1 exons 2, 3 and 4 gene sequences. the overall detection rate of sequencing and dHPLC screening methods was similar. Twenty mutations were detected in exon 2 and one mutation in exon 3 (c.231_232 dupGT) sequences of acute megakaryoblastic leukemia and transient leukemia samples. Four GATA1 mutations were newly described [c.155CG; c.156_178 del23 bp; c.29_30 del GG; c.182CA and c.151AT,c.153_162 del 10 bp). Out of four, three had single nucleotide change. in conclusion, our results indicate that dHPLC is an efficient and valuable tool for GATA1 mutational analysis.Inst Nacl Canc, Div Genet CPq, BR-20331050 Rio de Janeiro, BrazilHosp Darcy Vargas, Pediat Oncohematol Serv, São Paulo, BrazilSoc Oncol Bahia, Salvador, BrazilCtr Infantil Invest Hematol D Boldrini, São Paulo, BrazilHosp Santa Casa Misericordia, Pediat Oncohematol Serv, Itabuna, Bahia, BrazilHosp Martagao Gesteira, Pediat Oncohematol Serv, Salvador, BA, BrazilUniv Santa Maria Rio Grande Sul, Dept Hematol, Porto Alegre, RS, BrazilHosp Joana Gusmao Florianopolis, Pediat Oncohematol Serv, Santa Catarina, BrazilUniversidade Federal de São Paulo, Inst Oncol Pediat, São Paulo, BrazilHosp AC Camargo Fund Antonio Prudente, Pediat Oncohematol Serv, São Paulo, BrazilHosp Canc Cascavel, UOPECCAN, Cascavel, Parana, BrazilHosp Napoleao Laureano, Joao Pessoa, Paraiba, BrazilUFRJ, Inst Pediat & Puericultura Martagao Gesteira, Rio de Janeiro, Paraiba, BrazilHosp Serv Estado Rio de Janeiro, Dept Pediat, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Inst Oncol Pediat, São Paulo, BrazilWeb of Scienc

Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein.

International audienceIn humans, SHH haploinsufficiency results in holoprosencephaly (HPE), a defect in anterior midline formation. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a rare nucleotide variant located 460 kb upstream of SHH in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity-capture assay, we screened the SBE2 sequence for DNA-binding proteins and identified members of the Six3 and Six6 homeodomain family as candidate regulators of Shh transcription. Six3 showed reduced binding affinity for the mutant compared to the wild-type SBE2 sequence. Moreover, Six3 with HPE-causing alterations failed to bind and activate SBE2. These data suggest a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE