Alx4 is a paired-type homeodomain transcription factor that plays a key role in development, strongly expressed in the first branchial arch and craniofacial region. Pax3 also belongs to this family, and it displays a similar pattern of expression to that of Alx4. When Pax3 or Alx4 activity is lost individually, defects arise in an overlapping set of embryonic structures. In addition to their expression patterns, this suggests that these two factors may interact to play a role in normal murine development. We demonstrate an overlapping pattern of expression of Pax3 and Alx4 in the developing embryo and that Pax3 and Alx4 physically interact in vivo and in vitro. Pax3 and Alx4 can activate transcription from a P3 homeodomain consensus site, and preliminary analysis of mice null for both Pax3 and Alx4 show a novel mutant phenotype. We have therefore demonstrated a physical and genetic interaction between Pax3 and Alx4.

Alx4 is a paired-type homeodomain transcription factor that plays a key role in development, strongly expressed in the first branchial arch and craniofacial region. Pax3 also belongs to this family, and it displays a similar pattern of expression to that of Alx4. When Pax3 or Alx4 activity is lost individually, defects arise in an overlapping set of embryonic structures. In addition to their expression patterns, this suggests that these two factors may interact to play a role in normal murine development. We demonstrate an overlapping pattern of expression of Pax3 and Alx4 in the developing embryo and that Pax3 and Alx4 physically interact in vivo and in vitro. Pax3 and Alx4 can activate transcription from a P3 homeodomain consensus site, and preliminary analysis of mice null for both Pax3 and Alx4 show a novel mutant phenotype. We have therefore demonstrated a physical and genetic interaction between Pax3 and Alx4.

Aristaless-like homeobox 4 is a homeodomain transcription factor that has important functions during mouse development. A recent report demonstrated that Alx4 expression is required in periductal stromal cells in the mouse mammary gland for normal mammary morphogenesis. To test the hypothesis that ALX4 is expressed in the normal human breast, and this expression is altered in breast cancer, immunohistochemistry was performed on normal and breast cancer tissue and breast tissue microarrays. In the normal breast, ALX4 was expressed in stromal fibroblasts and luminal epithelial cells, but not in myoepithelial cells. Expression was lost in breast cancer in both cell compartments. Upon global demethylation induced by 5-aza-2’-deoxycytidine, normal and breast cancer cell lines expressed ALX4, suggesting that hypermethylation may repress expression of ALX4 during malignant transformation of the breast. These results demonstrate that ALX4 may be used as a biomarker for breast cancer, and may act as a tumour suppressor.

Aristaless-like homeobox 4 is a homeodomain transcription factor that has important functions during mouse development. A recent report demonstrated that Alx4 expression is required in periductal stromal cells in the mouse mammary gland for normal mammary morphogenesis. To test the hypothesis that ALX4 is expressed in the normal human breast, and this expression is altered in breast cancer, immunohistochemistry was performed on normal and breast cancer tissue and breast tissue microarrays. In the normal breast, ALX4 was expressed in stromal fibroblasts and luminal epithelial cells, but not in myoepithelial cells. Expression was lost in breast cancer in both cell compartments. Upon global demethylation induced by 5-aza-2’-deoxycytidine, normal and breast cancer cell lines expressed ALX4, suggesting that hypermethylation may repress expression of ALX4 during malignant transformation of the breast. These results demonstrate that ALX4 may be used as a biomarker for breast cancer, and may act as a tumour suppressor.

A displasia frontonasal (DFN) compreende quadros de aparência facial variável, sendo clinicamente caracterizada por dois ou mais dos seguintes sinais: hipertelorismo ocular com consequente alargamento da base nasal; fissura facial mediana afetando o nariz ou o nariz e lábio superior e, por vezes, o palato; fissura alar (uni ou bilateral); ponta nasal ausente; crânio anterior bífido oculto, e implantação em 'V' dos cabelos na fronte. A DFN pode ser vista como um defeito de desenvolvimento que pode ocorrer por si só ou como parte do quadro clínico de várias síndromes. A maioria dos casos de DFN é esporádica, e em raras circunstâncias foram observadas alterações cromossômicas em alguns indivíduos. Até o momento, quatro genes foram relacionados à patogênese molecular de algumas das síndromes com DFN, EFNB1, associado a uma forma de DFN ligada ao X e os genes ALX1, ALX3 e ALX4, todos associados a formas de DFN com herança autossômica recessiva. Embora esteja claro haver heterogeneidade etiológica, na maioria dos casos de DFN a causa não é conhecida, dificultando o adequado aconselhamento genético aos pacientes e seus familiares. Sendo assim, realizamos estudos com diferentes estratégias metodológicas buscando melhor compreender as possíveis causas genéticas da DFN. Ao todo foram analisados 10 pacientes: um caso familial de DFN leve com herança aparentemente autossômica dominante, um caso clinicamente sugestivo de mutação em ALX1, e oito casos de DFN associada a atraso de desenvolvimento com ou sem outras anomalias, dos quais um apresentava um rearranjo de novo aparentemente balanceado entre os cromossomos 4 e 12. Optamos por realizar sequenciamento dos genes previamente relacionados a fenótipos com DFN em todos os casos; para aqueles em que não foram detectadas mutações patogênicas, realizamos análise de variações de número de cópias (CNV) por microarray de polimorfismos de base única e, para o paciente com rearranjo cromossômico, realizamos o mapeamento do ponto de quebra por hibridação in situ fluorescente. Constatamos uma mutação em heterozigose no gene ALX4 co-segregando com o fenótipo do caso familial, sendo esta a primeira descrição de alteração em tal gene causando uma forma de DFN com herança dominante, e sugerimos pela primeira vez um mecanismo de dominância negativa. No caso sugestivo de mutação em ALX1, o diagnóstico foi confirmado através da identificação de uma mutação em homozigose neste gene do paciente; este caso consiste no 3o da literatura mundial e evidencia pela primeira vez que mutações em ALX1 não necessariamente levam a atraso de desenvolvimento ou deficiência intelectual. Os estudos citogenéticos e moleculares dos pontos de quebra do paciente com rearranjo cromossômico sugeriram os genes ARAP2 e CAND1 como possíveis responsáveis por seu quadro clínico, enquanto o estudo de CNVs nos indivíduos com DFN associada a atraso de desenvolvimento apontou os genes DNAJB12 e ENOX2 como possíveis candidatos para explicar o fenótipo de dois dos pacientes. É preciso que novos estudos sejam realizados a fim de melhor compreender o significado de tais achados e a real contribuição de cada gene para o desenvolvimento craniofacial humano e para a etiologia da DFN. Para os casos em que não foram identificadas alterações conclusivas no presente estudo, embora causas ambientais não possam ser descartadas, é preciso que seja investigada também a existência de fatores genéticos e epigenéticos não detectáveis pelas metodologias utilizadas, bem como a hipótese de mosaicismo somático. Nossos resultados, além de corroborarem o envolvimento dos genes ALX1 e ALX4 em fenótipos com DFN, sugerem também novos genes candidatos: ARAP2, CAND1, DNAJB12 e ENOX2 / Frontonasal dysplasia (FND) is a rare group of disorders that comprises cases with a variety of facial appearances, and is clinically characterized by two or more of the following signs: ocular hypertelorism with consequent broadening of the nasal root; median facial cleft affecting the nose and/or upper lip and palate; clefting of the alae nasi (uni or bilateral); lack of formation of the nasal tip; anterior cranium bifidum occultum; and a V-shaped frontal hairline. FND is a developmental defect that can occur alone or as part of several syndromes. Most cases of FND are sporadic, and in rare circumstances chromosomal alterations were observed in affected individuals. To date, four genes have been related to the molecular pathogenesis of some syndromes with DFN, one (EFNB1) is associated with an X-linked form while the 3 others (ALX1, ALX3 and ALX4) are associated with autosomal recessive forms. Although it is clear that FND is etiologic heterogeneous, the causative mechanism is unknown in most cases which makes it hard to give proper genetic counseling to patients and their families. In order to get new insights into the genetic mechanisms leading to FND, we performed studies with different methodologies. Altogether, 10 patients were analyzed: a familial case of a mild form of FND with an apparently autosomal dominant inheritance pattern, a case clinically suggestive of mutation in ALX1, and eight cases of FND associated with developmental delay with or without other anomalies, one of which with an apparently balanced de novo rearrangement between chromosomes 4 and 12. We chose to sequence the genes previously associated with FND phenotypes in all cases; for those in which pathogenic mutations were not detected, we conducted an analysis of copy number variations (CNV) by single nucleotide polymorphisms microarrays; for the patient with chromosomal rearrangement, we also mapped the breakpoints by using fluorescence in situ hybridization. We found a heterozygous mutation in ALX4 co-segregating with the phenotype of the familial case; this is the first description of mutation in this gene causing a form of FND with dominant inheritance pattern, and we suggested for the first time a dominant negative mechanism. In the case suggestive of mutation in ALX1, the diagnosis was confirmed by the identification of a homozygous mutation in this gene; this is the third case of the literature and shows for the first time that mutations in ALX1 are not necessarily related to developmental delay or intellectual disability. Breakpoints cytogenetic and molecular studies done with the patient with chromosomal rearrangement suggested ARAP2 and CAND1 genes as causative candidates for his condition, while the study of CNVs in individuals with FND associated with developmental delay pointed DNAJB12 and ENOX2 genes as possible candidates to explain the phenotypes of two of the patients. Further studies are necessary to better understand the significance of such findings and the actual contribution of each of these genes to human craniofacial development and the etiology of FND. Although environmental causes cannot be ruled out, it should also be investigated the existence of genetic and epigenetic factors as well as the possibility of somatic mosaicism, among the cases negative for the molecular approaches used in our study. Our results corroborate the involvement of ALX1 and ALX4 in FND phenotypes, and suggest new candidate genes: ARAP2, CAND1, DNAJB12 and ENOX2.

A displasia frontonasal (DFN) compreende quadros de aparência facial variável, sendo clinicamente caracterizada por dois ou mais dos seguintes sinais: hipertelorismo ocular com consequente alargamento da base nasal; fissura facial mediana afetando o nariz ou o nariz e lábio superior e, por vezes, o palato; fissura alar (uni ou bilateral); ponta nasal ausente; crânio anterior bífido oculto, e implantação em 'V' dos cabelos na fronte. A DFN pode ser vista como um defeito de desenvolvimento que pode ocorrer por si só ou como parte do quadro clínico de várias síndromes. A maioria dos casos de DFN é esporádica, e em raras circunstâncias foram observadas alterações cromossômicas em alguns indivíduos. Até o momento, quatro genes foram relacionados à patogênese molecular de algumas das síndromes com DFN, EFNB1, associado a uma forma de DFN ligada ao X e os genes ALX1, ALX3 e ALX4, todos associados a formas de DFN com herança autossômica recessiva. Embora esteja claro haver heterogeneidade etiológica, na maioria dos casos de DFN a causa não é conhecida, dificultando o adequado aconselhamento genético aos pacientes e seus familiares. Sendo assim, realizamos estudos com diferentes estratégias metodológicas buscando melhor compreender as possíveis causas genéticas da DFN. Ao todo foram analisados 10 pacientes: um caso familial de DFN leve com herança aparentemente autossômica dominante, um caso clinicamente sugestivo de mutação em ALX1, e oito casos de DFN associada a atraso de desenvolvimento com ou sem outras anomalias, dos quais um apresentava um rearranjo de novo aparentemente balanceado entre os cromossomos 4 e 12. Optamos por realizar sequenciamento dos genes previamente relacionados a fenótipos com DFN em todos os casos; para aqueles em que não foram detectadas mutações patogênicas, realizamos análise de variações de número de cópias (CNV) por microarray de polimorfismos de base única e, para o paciente com rearranjo cromossômico, realizamos o mapeamento do ponto de quebra por hibridação in situ fluorescente. Constatamos uma mutação em heterozigose no gene ALX4 co-segregando com o fenótipo do caso familial, sendo esta a primeira descrição de alteração em tal gene causando uma forma de DFN com herança dominante, e sugerimos pela primeira vez um mecanismo de dominância negativa. No caso sugestivo de mutação em ALX1, o diagnóstico foi confirmado através da identificação de uma mutação em homozigose neste gene do paciente; este caso consiste no 3o da literatura mundial e evidencia pela primeira vez que mutações em ALX1 não necessariamente levam a atraso de desenvolvimento ou deficiência intelectual. Os estudos citogenéticos e moleculares dos pontos de quebra do paciente com rearranjo cromossômico sugeriram os genes ARAP2 e CAND1 como possíveis responsáveis por seu quadro clínico, enquanto o estudo de CNVs nos indivíduos com DFN associada a atraso de desenvolvimento apontou os genes DNAJB12 e ENOX2 como possíveis candidatos para explicar o fenótipo de dois dos pacientes. É preciso que novos estudos sejam realizados a fim de melhor compreender o significado de tais achados e a real contribuição de cada gene para o desenvolvimento craniofacial humano e para a etiologia da DFN. Para os casos em que não foram identificadas alterações conclusivas no presente estudo, embora causas ambientais não possam ser descartadas, é preciso que seja investigada também a existência de fatores genéticos e epigenéticos não detectáveis pelas metodologias utilizadas, bem como a hipótese de mosaicismo somático. Nossos resultados, além de corroborarem o envolvimento dos genes ALX1 e ALX4 em fenótipos com DFN, sugerem também novos genes candidatos: ARAP2, CAND1, DNAJB12 e ENOX2 / Frontonasal dysplasia (FND) is a rare group of disorders that comprises cases with a variety of facial appearances, and is clinically characterized by two or more of the following signs: ocular hypertelorism with consequent broadening of the nasal root; median facial cleft affecting the nose and/or upper lip and palate; clefting of the alae nasi (uni or bilateral); lack of formation of the nasal tip; anterior cranium bifidum occultum; and a V-shaped frontal hairline. FND is a developmental defect that can occur alone or as part of several syndromes. Most cases of FND are sporadic, and in rare circumstances chromosomal alterations were observed in affected individuals. To date, four genes have been related to the molecular pathogenesis of some syndromes with DFN, one (EFNB1) is associated with an X-linked form while the 3 others (ALX1, ALX3 and ALX4) are associated with autosomal recessive forms. Although it is clear that FND is etiologic heterogeneous, the causative mechanism is unknown in most cases which makes it hard to give proper genetic counseling to patients and their families. In order to get new insights into the genetic mechanisms leading to FND, we performed studies with different methodologies. Altogether, 10 patients were analyzed: a familial case of a mild form of FND with an apparently autosomal dominant inheritance pattern, a case clinically suggestive of mutation in ALX1, and eight cases of FND associated with developmental delay with or without other anomalies, one of which with an apparently balanced de novo rearrangement between chromosomes 4 and 12. We chose to sequence the genes previously associated with FND phenotypes in all cases; for those in which pathogenic mutations were not detected, we conducted an analysis of copy number variations (CNV) by single nucleotide polymorphisms microarrays; for the patient with chromosomal rearrangement, we also mapped the breakpoints by using fluorescence in situ hybridization. We found a heterozygous mutation in ALX4 co-segregating with the phenotype of the familial case; this is the first description of mutation in this gene causing a form of FND with dominant inheritance pattern, and we suggested for the first time a dominant negative mechanism. In the case suggestive of mutation in ALX1, the diagnosis was confirmed by the identification of a homozygous mutation in this gene; this is the third case of the literature and shows for the first time that mutations in ALX1 are not necessarily related to developmental delay or intellectual disability. Breakpoints cytogenetic and molecular studies done with the patient with chromosomal rearrangement suggested ARAP2 and CAND1 genes as causative candidates for his condition, while the study of CNVs in individuals with FND associated with developmental delay pointed DNAJB12 and ENOX2 genes as possible candidates to explain the phenotypes of two of the patients. Further studies are necessary to better understand the significance of such findings and the actual contribution of each of these genes to human craniofacial development and the etiology of FND. Although environmental causes cannot be ruled out, it should also be investigated the existence of genetic and epigenetic factors as well as the possibility of somatic mosaicism, among the cases negative for the molecular approaches used in our study. Our results corroborate the involvement of ALX1 and ALX4 in FND phenotypes, and suggest new candidate genes: ARAP2, CAND1, DNAJB12 and ENOX2.