Genome-Wide Profiling of p63 DNA–Binding Sites Identifies an Element that Regulates Gene Expression during Limb Development in the 7q21 SHFM1 Locus

Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA–binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP–seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes.     

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile α-motif (SAM) domain (residues 505–579), a region crucial to explaining the human genetic disease ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of free-dom of individual residues and discussion of the possible causes for the pathology.     

Partial monosomy of the long arm of chromosome 16: A distinct clinical entity?

Summary A 7-month-old male child with a de novo, seemingly belanced reciprocal 5p/16q translocation and karyotype 46,XY,t(5;16) (p14;q21), resulting from a maternal meiotic error, is described. The clinical findings in this patient are strikingly similar to those in the only patient with partial deletion 16q hitherto described, [del(16)(q21)], indicating that during the 5p/16q rearrangement, 16q material was lost and suggesting that partial or total deletion of the long arm of chromosome 16 distal to band q21 is accompanied by a distinct clinical phenotype.     

Molecular cytogenetic characterization of 17 rob(13q14q) Robertsonian translocations by FISH, narrowing the region containing the breakpoints.

We have characterized 17 rob(13q14q) Robertsonian translocations, using six molecular probes that hybridize to the repetitive sequences of the centromeric and shortarm regions of the five acrocentric chromosomes by FISH. The rearrangements include six de novo rearrangements and the chromosomally normal parents, five maternally and three paternally inherited translocations, and three translocations of unknown origin. The D21Z1/D13Z1 and D14Z1/D22Z1 centromeric alpha-satellite DNA probes showed all rob(13q14q) chromosomes to be dicentric. The rDNA probes did not show hybridization on any of the 17 cases studied. The pTRS-47 satellite III DNA probe specific for chromosomes 14 and 22 was retained around the breakpoints in all cases. However, the pTRS-63 satellite III DNA probe specific for chromosome 14 did not show any signals on the translocation chromosomes examined. In 16 of 17 translocations studied, strong hybridization signals on the translocations were detected with the pTRI-6 satellite I DNA probe specific for chromosome 13. All parents of the six de novo rob(13q14q), including one whose pTRI-6 sequence was lost, showed strong positive hybridization signals on each pair of chromosomes 14 and 13, with pTRS-47, pTRS-63, and pTRI-6. Therefore, the translocation breakpoints in the majority of rob(13q14q) are between the pTRS-47 and pTRS-63 sequences in the p11 region of chromosome 14 and between the pTRI-6 and rDNA sequences within the p11 region of chromosome 13.     

Wnt9b is the mutated gene involved in multifactorial nonsyndromic cleft lip with or without cleft palate in A/WySn mice, as confirmed by a genetic complementation test

BACKGROUND: Nonsyndromic cleft lip (CL) with or without cleft palate (CLP) is a common human birth defect with complex genetic etiology. One of the unidentified genes maps to chromosome 17q21. A mouse strain, A/WySn, has CLP with complex genetic etiology that models the human defect, and 1 of its causative genes, clf1, maps to a region homologous to human 17q21. Extensive studies of the candidate region pointed to a novel insertion of an IAP transposon 3' from the gene Wnt9b as the clf1 mutation. Independently a recessive knockout mutation of Wnt9b (Wnt9b-) was reported to cause a lethal syndrome that includes some CLP. METHODS: A standard genetic test of allelism between clf1 and the Wnt9b- mutation was done. A total of 83 F1 embryos at gestation day 14 (GD 14) from Wnt9b-/+ males crossed with A/WySn females, and 79 BC1 GD 14 embryos from F1 Wnt9b-/clf1 males back-crossed to A/WySn females were observed for CL. Embryo genotypes at clf1 and Wnt9b were obtained from DNA markers. Genotypes for a second unlinked modifier locus from A/WySn, clf2, were similarly obtained. RESULTS: The compound mutant embryos (Wnt9b-/clf1) had high frequencies of CL: 27% in the F1 and 63% in the BC1. The clf2 modifier gene was found to have 3 alleles segregating in this study and to strongly influence the penetrance of CL in the compound mutant. CONCLUSIONS: The noncomplementation of clf1 and Wnt9b- confirms that clf1 is a mutation of the Wnt9b gene. The homologous human WNT9B gene and 3' conserved noncoding region should be examined for a role in human nonsyndromic CLP.     

A patient with Down syndrome with a de novo derivative chromosome 21

Pure partial trisomy of chromosome 21 is a rare event. The patients with this aberration are very important for setting up precise karyotype-phenotype correlations particularly in Down syndrome phenotype. We present here a patient with Down syndrome with a de novo derivative chromosome 21. Karyotype of the patient was designated as 46,XY,der(21)(p13)dup(21)(q11.2q21.3)dup(21)(q22.2q22.3) with regard to cytogenetic, FISH and array-CGH analyses. Non-continuous monosomic, disomic and trisomic chromosomal segments through the derivative chromosome 21 were detected by array-CGH analysis. STR analyses revealed maternal origin of the de novo derivative chromosome 21. The dual-specificity tyrosine (Y)-phosphorylation regulated kinase 1A (DYRK1A) and Down Syndrome Critical Region 1 (DSCR1) genes that are located in Down syndrome critical region, are supposed to be responsible for most of the clinical findings of Down syndrome. However, our patient is the first patient with Down syndrome whose clinical findings were provided in detail, with a de novo derivative chromosome 21 resulting from multiple chromosome breaks excluding DYRK1A and DSCR1 gene regions.     

A novel c.1037C > G (p.Ala346Gly) mutation in TP63 as cause of the ectrodactyly-ectodermal dysplasia and cleft lip/palate (EEC) syndrome

Ectrodactyly – ectodermal dysplasia and cleft lip/palate (EEC) syndrome (OMIM 604292) is a rare disorder determined by mutations in the TP63 gene. Most cases of EEC syndrome are associated to mutations in the DNA binding domain (DBD) region of the p63 protein. Here we report on a three-generation Brazilian family with three individuals (mother, son and grandfather) affected by EEC syndrome, determined by a novel mutation c.1037C > G (p.Ala346Gly). The disorder in this family exhibits a broad spectrum of phenotypes: two individuals were personally examined, one presenting the complete constellation of EEC syndrome manifestations and the other presenting an intermediate phenotype; the third affected, a deceased individual not examined personally and referred to by his daughter, exhibited only the split-hand/foot malformation (SHFM). Our findings contribute to elucidate the complex phenotype-genotype correlations in EEC syndrome and other related TP63-mutation syndromes. The possibility of the mutation c.1037C > G being related both to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM is also raised by the findings here reported.     

Novel homozygous mutations in the WNT10B gene underlying autosomal recessive split hand/foot malformation in three consanguineous families

Split-hand/split-foot malformation (SHFM), representing variable degree of median clefts of hands and feet, is a genetically heterogeneous group of limb malformations with seven loci mapped on different human chromosomes. However, only 3 genes (TP63, WNT10B, DLX5) for the seven loci have been identified.     

Identification of DNA sequences flanking the breakpoint of human t(14q21q) Robertsonian translocations.

We have employed molecular probes and in situ hybridization to investigate the DNA sequences flanking the breakpoint of a group of t(14q21q) Robertsonian translocations. In all the families studied, the probands were patients with Down syndrome who carried a de novo t(14q21q) translocation. The DNA probes used were two alphoid sequences, alphaRI and alphaXT, which are specific for the centromeres of chromosomes 13 and 21 and of chromosomes 14 and 22, respectively; a satellite III sequence, pTRS-47, which is specific for the proximal p11 region of chromosomes 14 and 22; and a newly defined satellite III DNA, pTRS-63, which is specific for the distal p11 region of chromosome 14. The two alphoid probes detected approximately the same amount of autoradiographic signal on the translocated chromosomes as was expected for chromosomes 14 and 21 of the originating parent, suggesting that there has been no loss of these centromeric sequences during the translocation events. Results with the two satellite III probes indicated that the domain corresponding to pTRS-47 was retained in the translocated chromosomes, whereas the domain for pTRS-63 was lost. These results have allowed us to place the translocation breakpoint between the pTRS-47 and pTRS-63 domains within the p11 region of chromosome 14.     

Generation of a complete set of human telomeric band painting probes by chromosome microdissection

Chromosomal rearrangements involving telomeric bands have been frequently detected in many malignancies and congenital diseases. To develop a useful tool to study chromosomal rearrangements within the telomeric band effectively and accurately, a whole set of telomeric band painting probes (TBP) has been generated by chromosome microdissection. The intensity and specificity of these TBPs have been tested by fluorescence in situ hybridization and all TBPs showed strong and specific signals to target regions. TBPs of 6q and 17p were successfully used to detect the loss of the terminal band of 6q in a hepatocellular carcinoma cell line and a complex translocation involving the 17p terminal band in a melanoma cell line. Meanwhile, the TBP of 21q was used to detect a de novo translocation, t(12;21), and the breakpoint at 21q was located at 21q22.2. Further application of these TBPs should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements involving telomeric bands.     

Special AT-rich binding protein-2 (SATB2) differentially affects disease-causing p63 mutant proteins

p63, a p53 family member, is critical for proper skin and limb development and directly regulates gene expression in the ectoderm. Mice lacking p63 exhibit skin and craniofacial defects including cleft palate. In humans p63 mutations are associated with several distinct developmental syndromes. p63 sterile-α-motif domain, AEC (ankyloblepharon-ectodermal dysplasia-clefting)-associated mutations are associated with a high prevalence of orofacial clefting disorders, which are less common in EEC (ectrodactyly-ectodermal dysplasia-clefting) patients with DNA binding domain p63 mutations. However, the mechanisms by which these mutations differentially influence p63 function remain unclear, and interactions with other proteins implicated in craniofacial development have not been identified. Here, we show that AEC p63 mutations affect the ability of the p63 protein to interact with special AT-rich binding protein-2 (SATB2), which has recently also been implicated in the development of cleft palate. p63 and SATB2 are co-expressed early in development in the ectoderm of the first and second branchial arches, two essential sites where signaling is required for craniofacial patterning. SATB2 attenuates p63-mediated gene expression of perp (p53 apoptosis effector related to PMP-22), a critical downstream target gene during development, and specifically decreases p63 perp promoter binding. Interestingly, AEC but not EEC p63 mutations affect the ability of p63 to interact with SATB2 and the inhibitory effects of SATB2 on p63 transactivation of perp are most pronounced for AEC-associated p63 mutations. Our findings reveal a novel gain-of-function property of AEC-causing p63 mutations and identify SATB2 as the first p63 binding partner that differentially influences AEC and EEC p63 mutant proteins.     

人体乳腺癌细胞系Bcap－37和MCF－7的细胞遗传学研究

应用低温同步法与秋水酰胺处理,对人体乳腺癌细胞系Ｂｃａｐ－３７和ＭＣＦ－７的中期及早中期细胞进行Ｇ－显带分析。研究表明,Ｂｃａｐ－３７细胞染色体众数为６３,可识别其结构的标记染色体１７条;ＭＣＦ－７细胞染色体众数为５６,可识别其结构的标记染色体１３条。结合文献报道以及本研究结果显示,乳腺癌中最常涉及到第１、３、５、７、１１、１３和１７号染色体结构及数目的异常,染色体断裂点１ｐ１１（１ｑ１１）、１ｐ１３、３ｐ２１、３ｑ１１、５ｑ１１、６ｑ１３、６ｑ２３、７ｑ２２、１１ｐ１３和１１ｐ１５也经常涉及;它们可能与癌相关基因的激活和抗癌基因的丢失有关,从而在乳腺癌发生发展中起一定作用。     

Partial monosomy 21 (q11.2→q21.3) combined with 3p25.3→pter monosomy due to an unbalanced translocation in a patient presenting dysmorphic features and developmental delay

We describe a female patient of 1 year and 5 months-old, referred for genetic evaluation due to neuropsychomotor delay, hearing impairment and dysmorphic features. The patient presents a partial chromosome 21 monosomy (q11.2→q21.3) in combination with a chromosome 3p terminal monosomy (p25.3→pter) due to an unbalanced de novo translocation. The translocation was confirmed by fluorescence in situ hybridization (FISH) and the breakpoints were mapped with high resolution array. After the combined analyses with these techniques the final karyotype was defined as 45,XX,der(3)t(3;21)(p25.3;q21.3)dn,-21.ish der(3)t(3;21)(RP11-329A2-,RP11-439F4-,RP11-95E11-,CTB-63H24 +).arr 3p26.3p25.3(35,333-10,888,738)) × 1,21q11.2q21.3(13,354,643-27,357,765) × 1. Analysis of microsatellite DNA markers pointed to a paternal origin for the chromosome rearrangement. This is the first case described with a partial proximal monosomy 21 combined with a 3p terminal monosomy due to a de novo unbalanced translocation.     

Genomic imbalances in 61 renal cancers from the proximal tubulus detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to characterize 61 primary renal cell carcinomas derived histogenetically from the proximal tubulus. The tumor samples comprised 46 clear-cell renal cell carcinomas (ccRCCs) and 15 papillary renal cell carcinomas (pRCCs). Changes in the copy number of entire chromosomes or subregions were detected in 56 tumors (92%). In ccRCCs, losses of chromosome 3 or 3p (63%); 14q (30%); 9 (26%); 1 and 6 or 6q (17% each); 4 and 8 or 8p (15% each); 22 (11%); 2 or 2q and 19 (9% each); 7q, 10, 16, 17p, 18, and Y (7% each); and 5, 11, 13, 15, and 21 (4% each) were detected. Most frequent genomic gains in ccRCC were found on chromosome 5 (63%); 7 (35%); 1 or 1q (33%); 2q (24%); 8 or 8q, 12, and 20 (20% each); 3q (17%); 16 (15%); 19 (13%); 6 and 17 or 17q (11% each); and 4, 10, 11, 21, and Y (9% each). In pRCCs, gains in the copy number of chromosomes 7 and 17 (7/15, each) and 16 and 20 (6/15, each) were frequent. One pRCC showed amplification of subchromosome regions 2q22-->q33, 16q, 17q and the entire X chromosome. In pRCC, losses were less frequently seen than gains. Losses of chromosomes 1, 14, 15, and Y (3/15 each) and 2, 4, 6, and 13 (2/15 each) were observed. In ccRCCs, statistical evaluation revealed significant correlations of chromosomal imbalances with tumor stage and grade, i.e., a gain in copy number of chromosome 5 correlated positively with low tumor grade, whereas a gain of chromosomes 10 and 17 correlated positively with high tumor grade. Furthermore, loss of chromosome 4 correlated positively with high tumor stage.     

A digenic cause of cleft lip in A-strain mice and definition of candidate genes for the two loci

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate, CL(P), is a common human birth defect with a complex unknown genetic cause. The mouse model is the "A/-" strains. Our previous studies mapped two loci: clf1 on Chr11 and clf2 on Chr13--with a strong genetic maternal effect on the level of risk. Here we test the hypothesis that CL(P) is digenic and identify candidate genes for clf1 and clf2. METHODS: We observed E14 CL(P) frequencies in backcross (BC1) embryos from a new cross of A/WySn to AXB-4/Pgn and from test crosses of three new "congenic RI" lines. Using new polymorphic markers from genes and our mapping panels of segregants and RI strains, we identified the candidate genes for clf1 and clf2. We sequenced the coding region of Ptch in A/WySn cDNA. RESULTS: Seventy new BC1 CL(P) segregants (4%) were obtained, as predicted. All three new congenic RI lines homozygous for both clf1 and clf2 had A/WySn-level CL(P) frequencies (10-30%) in test crosses. The clf1 region contains 10 known genes (Arf2, Cdc27, Crhr1, Gosr2, Itgb3, Mapt, Myl4, Nsf, Wnt3, and Wnt9b). The clf2 region contains 17 known genes with human orthologs. Both regions contain additional potential genes. No causal mutation in Ptch coding sequence was found. CONCLUSIONS: In A-strain mice, nonsyndromic CL(P) is digenic, suggesting that nonsyndromic human CL(P) may also be digenic. The orthologous human genes are on 17q (clf1) and 9q, 8q and 5p (clf2), and good candidate genes are WNT3 or WNT9B (17q), and PTCH (9q) or MTRR (5p).     

Genome-wide linkage in a highly consanguineous pedigree reveals two novel loci on chromosome 7 for non-syndromic familial Premature Ovarian Failure

Background

The human condition known as Premature Ovarian Failure (POF) is characterized by loss of ovarian function before the age of 40. A majority of POF cases are sporadic, but 10–15% are familial, suggesting a genetic origin of the disease. Although several causal mutations have been identified, the etiology of POF is still unknown for about 90% of the patients.

Methodology/Principal Findings

We report a genome-wide linkage and homozygosity analysis in one large consanguineous Middle-Eastern POF-affected family presenting an autosomal recessive pattern of inheritance. We identified two regions with a LOD_max of 3.26 on chromosome 7p21.1-15.3 and 7q21.3-22.2, which are supported as candidate regions by homozygosity mapping. Sequencing of the coding exons and known regulatory sequences of three candidate genes (DLX5, DLX6 and DSS1) included within the largest region did not reveal any causal mutations.

Conclusions/Significance

We detect two novel POF-associated loci on human chromosome 7, opening the way to the identification of new genes involved in the control of ovarian development and function.     

Analysis of susceptibility polymorphisms for nonsyndromic cleft lip with or without cleft palate in the Brazilian population

Background : Although genome‐wide association studies have identified several susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P) in populations around the world, the role of most loci is unknown in the highly heterogeneous Brazilian population. Methods : To determine the association of 7 markers that showed genome‐wide significant association in Brazilians with NSCL/P, we conducted a structured association study conditioned upon the individual ancestry proportions to evaluate markers at 1p36 (rs742071), 2p21 (rs7590268), 3p11.1 (rs7632427), 8q21.3 (rs12543318), 13q31.1 (rs8001641), 15q22.2 (rs1873147), and 17q22 (rs227731) in 505 patients with NSCL/P and 594 healthy controls recruited from 2 different geographical regions of Brazil. The polymorphisms were genotyped by TaqMan 5′‐exonuclease allelic discrimination assay, and each sample was independently typed for 40 biallelic short insertion/deletion markers to characterize the genomic ancestry. Results : After Bonferroni correction for multiple tests, significant associations with NSCL/P were observed for rs742071, rs1873147, and rs227731. However, the frequency of the risk alleles varied between the geographical regions, according to the proportions of European and African genomic ancestry. The group enriched by European ancestry showed significant association with rs227731 (p = 0.001), whereas the group with high African ancestry was significantly associated with rs1873147 polymorphism (p = 0.005). The significant association with rs742071 was only detected in the combined sample (p = 0.005). Conclusion : The findings of the present study revealed the associations of 1p36 (rs742071), 15q22 (rs1873147), and 17p22 (rs227731) with NSCL/P in the Brazilian population, and further confirmed that the genetic heterogeneity of NSCL/P may be related to the different ethnic background of the affected individuals. Birth Defects Research (Part A) 100:36–42, 2014. © 2013 Wiley Periodicals, Inc.