Haploinsufficiency of SF3B4, a component of the pre-mRNA spliceosomal complex, causes Nager syndrome

Nager syndrome, first described more than 60 years ago, is the archetype of a class of disorders called the acrofacial dysostoses, which are characterized by craniofacial and limb malformations. Despite intensive efforts, no gene for Nager syndrome has yet been identified. In an international collaboration, FORGE Canada and the National Institutes of Health Centers for Mendelian Genomics used exome sequencing as a discovery tool and found that mutations in SF3B4, a component of the U2 pre-mRNA spliceosomal complex, cause Nager syndrome. After Sanger sequencing of SF3B4 in a validation cohort, 20 of 35 (57%) families affected by Nager syndrome had 1 of 18 different mutations, nearly all of which were frameshifts. These results suggest that most cases of Nager syndrome are caused by haploinsufficiency of SF3B4. Our findings add Nager syndrome to a growing list of disorders caused by mutations in genes that encode major components of the spliceosome and also highlight the synergistic potential of international collaboration when exome sequencing is applied in the search for genes responsible for rare Mendelian phenotypes.     

Clinical and mutation data in 12 patients with the clinical diagnosis of Nager syndrome

Nager syndrome (MIM #154400) is the best-known preaxial acrofacial dysostosis, mainly characterized by craniofacial and preaxial limb anomalies. The craniofacial abnormalities mainly consist of downslanting palpebral fissures, malar hypoplasia, micrognathia, external ear anomalies, and cleft palate. The preaxial limb defects are characterized by radial and thumb hypoplasia or aplasia, duplication of thumbs and proximal radioulnar synostosis. Haploinsufficiency of SF3B4 (MIM *605593), which encodes SAP49, a component of the pre-mRNA spliceosomal complex, has recently been identified as the underlying cause of Nager syndrome. In our study, we performed exome sequencing in two and Sanger sequencing of SF3B4 in further ten previously unreported patients with the clinical diagnosis of Nager syndrome, including one familial case. We identified heterozygous SF3B4 mutations in seven out of twelve patients. Four of the seven mutations were shown to be de novo; in three individuals, DNA of both parents was not available. No familial mutations were discovered. Three mutations were nonsense, three were frameshift mutations and one T > C transition destroyed the translation start signal. In three of four SF3B4 negative families, EFTUD2 was analyzed, but no pathogenic variants were identified. Our results indicate that the SF3B4 gene is mutated in about half of the patients with the clinical diagnosis of Nager syndrome and further support genetic heterogeneity for this condition.     

Acrofacial Dysostosis,Cincinnati Type,a Mandibulofacial Dysostosis Syndrome with Limb Anomalies,Is Caused by POLR1A Dysfunction

We report three individuals with a cranioskeletal malformation syndrome that we define as acrofacial dysostosis, Cincinnati type. Each individual has a heterozygous mutation in POLR1A, which encodes a core component of RNA polymerase 1. All three individuals exhibit varying degrees of mandibulofacial dysostosis, and two additionally have limb anomalies. Consistent with this observation, we discovered that polr1a mutant zebrafish exhibited cranioskeletal anomalies mimicking the human phenotype. polr1a loss of function led to perturbed ribosome biogenesis and p53-dependent cell death, resulting in a deficiency of neural-crest-derived skeletal precursor cells and consequently craniofacial anomalies. Our findings expand the genotypic and phenotypic heterogeneity of congenital acrofacial disorders caused by disruption of ribosome biogenesis.     

Mutations in SOX9, the Gene Responsible for Campomelic Dysplasia and Autosomal Sex Reversal

Campomelic dysplasia (CD) is a skeletal malformation syndrome frequently accompanied by 46,XY sex reversal. A mutation-screening strategy using SSCP was employed to identify mutations in SOX9, the chromosome 17q24 gene responsible for CD and autosomal sex reversal in man. We have screened seven CD patients with no cytologically detectable chromosomal aberrations and two CD patients with chromosome 17 rearrangements for mutations in the entire open reading frame of SOX9. Five different mutations have been identified in six CD patients: two missense mutations in the SOX9 putative DNA binding domain (high mobility group, or HMG, box); three frameshift mutations and a splice-acceptor mutation. An identical frameshift mutation is found in two unrelated 46,XY patients, one exhibiting a male phenotype and the other displaying a female phenotype (XY sex reversal). All mutations found affect a single allele, which is consistent with a dominant mode of inheritance. No mutations were found in the SOX9 open reading frame of two patients with chromosome 17q rearrangements, suggesting that the translocations affect SOX9 expression. These findings are consistent with the hypothesis that CD results from haploinsufficiency of SOX9.     

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.     

Transcriptional Factor DLX3 Promotes the Gene Expression of Enamel Matrix Proteins during Amelogenesis

Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.     

A novel heterozygous deletion in the EVC2 gene causes Weyers acrofacial dysostosis

Weyers acrofacial dysostosis (MIM 193530) is an autosomal dominant disorder clinically characterized by mild short stature, postaxial polydactyly, nail dystrophy and dysplastic teeth. Ellis–van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive disorder with a similar, but more severe phenotype. Mutations in the EVC have been identified in both syndromes. However, the EVC mutations only occur in a small proportion of EvC patients. Recently, mutations in a new gene, EVC2, were found to be associated with other EvC cases. The EVC and EVC2 are located close to each other in a head-to-head configuration and may be functionally related. In this study, we report identification of a novel heterozygous deletion in the EVC2 that is responsible for autosomal dominant Weyers acrofacial dysostosis in a large Chinese family. This constitutes the first report of Weyers acrofacial dysostosis caused by this gene. Hence, the spectrum of malformation syndromes due to EVC2 mutations is further extended. Our data provides conclusive evidence that Weyers acrofacial dysostosis and EvC syndrome are allelic and genetically heterogeneous conditions. X. Ye and G. Song contributed equally to this work     

A human homeotic transformation resulting from mutations in PLCB4 and GNAI3 causes auriculocondylar syndrome

Auriculocondylar syndrome (ACS) is a rare, autosomal-dominant craniofacial malformation syndrome characterized by variable micrognathia, temporomandibular joint ankylosis, cleft palate, and a characteristic “question-mark” ear malformation. Careful phenotypic characterization of severely affected probands in our cohort suggested the presence of a mandibular patterning defect resulting in a maxillary phenotype (i.e., homeotic transformation). We used exome sequencing of five probands and identified two novel (exclusive to the patient and/or family studied) missense mutations in PLCB4 and a shared mutation in GNAI3 in two unrelated probands. In confirmatory studies, three additional novel PLCB4 mutations were found in multigenerational ACS pedigrees. All mutations were confirmed by Sanger sequencing, were not present in more than 10,000 control chromosomes, and resulted in amino-acid substitutions located in highly conserved protein domains. Additionally, protein-structure modeling demonstrated that all ACS substitutions disrupt the catalytic sites of PLCB4 and GNAI3. We suggest that PLCB4 and GNAI3 are core signaling molecules of the endothelin-1-distal-less homeobox 5 and 6 (EDN1-DLX5/DLX6) pathway. Functional studies demonstrated a significant reduction in downstream DLX5 and DLX6 expression in ACS cases in assays using cultured osteoblasts from probands and controls. These results support the role of the previously implicated EDN1-DLX5/6 pathway in regulating mandibular specification in other species, which, when disrupted, results in a maxillary phenotype. This work defines the molecular basis of ACS as a homeotic transformation (mandible to maxilla) in humans.     

Chronic constipation recognized as a sign of a SOX10 mutation in a patient with Waardenburg syndrome

Waardenburg syndrome is characterized by hearing loss, pigmentation abnormalities, dysmorphologic features, and neurological phenotypes. Waardenburg syndrome consists of four distinct subtypes, and SOX10 mutations have been identified in type II and type IV. Type IV differs from type II owing to the presence of Hirschsprung disease. We identified a de novo nonsense mutation in SOX10 (p.G39X) in a female pediatric patient with Waardenburg syndrome with heterochromia iridis, profound bilateral sensorineural hearing loss, inner ear malformations, and overall hypopigmentation of the hair without dystopia canthorum. This patient has experienced chronic constipation since she was a neonate, but anorectal manometry showed a normal anorectal reflex. Chronic constipation in this patient was likely to be a consequence of a mild intestinal disorder owing to the SOX10 mutation, and this patient was considered to have a clinical phenotype intermediate between type II and type IV of the syndrome. Chronic constipation may be recognized as indicative of a SOX10 mutation in patients with Waardenburg syndrome.     

Identification and functional analysis of a novel mutation in the SOX10 gene associated with Waardenburg syndrome type IV

Waardenburg syndrome type IV (WS4) is a rare genetic disorder, characterized by auditory–pigmentary abnormalities and Hirschsprung disease. Mutations of the EDNRB gene, EDN3 gene, or SOX10 gene are responsible for WS4. In the present study, we reported a case of a Chinese patient with clinical features of WS4. In addition, the three genes mentioned above were sequenced in order to identify whether mutations are responsible for the case. We revealed a novel nonsense mutation, c.1063C>T (p.Q355*), in the last coding exon of SOX10. The same mutation was not found in three unaffected family members or 100 unrelated controls. Then, the function and mechanism of the mutation were investigated in vitro. We found both wild-type (WT) and mutant SOX10 p.Q355* were detected at the expected size and their expression levels are equivalent. The mutant protein also localized in the nucleus and retained the DNA-binding activity as WT counterpart; however, it lost its transactivation capability on the MITF promoter and acted as a dominant-negative repressor impairing function of the WT SOX10.     

Isolation and identification of homeobox genes from the human placenta including a novel member of the Distal-less family,DLX4

We have carried out a DNA binding site screen of a 32-week human placental cDNA library using a consensus homeodomain binding site as a probe. This study represents the first library screen carried out to isolate homeobox genes from the human placenta. We have shown that three homeobox genes known to be expressed in the embryo, HB24, GAX and MSX2 are also expressed in the placenta. We have also identified a novel homeobox gene, DLX4, that shows 85% sequence identity with the homeodomain encoded by the Drosophila Distal-less (Dll) gene. DLX4 therefore represents a new member of the Distal-less family of homeobox genes. This is the first evidence that members of the Distal-less family of homeobox genes are expressed in the placenta. Using fluorescence in situ hybridisation (FISH), DLX4 has been assigned to human chromosome 17q21–q22. This places DLX4 in the same region of chromosome 17 as another member of the Distal-less family, DLX3 (Scherer et al., 1995), and the HOX-B homeobox gene cluster (Acampora et al., 1989; Boncinelli et al., 1991). Members of the Distal-less family (DLX1 and DLX2; DLX5 and DLX6) are found as closely linked pairs on human chromosomes (Simeone et al., 1994). We predict that DLX3 and DLX4 are closely linked and have arisen through gene duplication and divergence from a common ancestral precursor.     

Genome-wide DNA methylation profiling of chronic lymphocytic leukemia allows identification of epigenetically repressed molecular pathways with clinical impact

We performed a genome-wide analysis of aberrant DNA methylation in chronic lymphocytic leukemia (CLL) using methylated CpG island amplification (MCA) coupled with a promoter microarray. We identified 280 potential targets of aberrant DNA methylation in CLL. These genes were located more frequently in chromosomes 19 (16%, p = 0.001), 16 (11%, p = 0.001), 17 (10%, p = 0.02) and 11 (9%, p = 0.02) and could be grouped in several functional networks. Methylation status was confirmed for 22 of these genes (SOX11, DLX1, FAM62C, SOX14, RSPO1, ADCY5, HAND2, SPOCK, MLL, ING1, PRIMA1, BCL11B, LTBP2, BNC1, NR2F2, SALL1, GALGT2, LHX1, DLX4, KLK10, TFAP2 and APP) in 78 CLL patients by pyrosequencing. As a proof of principle, we analyzed the expression of 2 genes, PRIMA1 and APP, in primary cells and of GALGT2, TFAP2C and PRIMA1 in leukemia cells. There was an inverse association between methylation and gene expression. This could be reversed by treatment with 5-aza-2′-deoxycytidine in cell lines. Treatment in a clinical trial with 5-azacitidine resulted in decreased methylation of LINE, DLX4 and SALL1 in the peripheral blood B-cells of patients with CLL. IgV_H mutational status or ZAP-70 expression were not associated with specific methylation profiles. By multivariate analysis, methylation of LINE and APP was associated with shorter overall survival (p = 0.045 and 0.0035, respectively). This study demonstrates that aberrant DNA methylation is common and has potential prognostic and therapeutic value in CLL.Key words: chronic lymphocytic leukemia, DNA methylation, MCA/promoter microarray, epigenetics     

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.     

Mutations in the Testis-Specific Enhancer of SOX9 in the SRY Independent Sex-Determining Mechanism in the Genus Tokudaia

SRY (sex-determining region Y) is widely conserved in eutherian mammals as a sex-determining gene located on the Y chromosome. SRY proteins bind to the testis-specific enhancer of SOX9 (TES) with SF1 to upregulate SOX9 expression in undifferentiated gonads of XY embryos of humans and mice. The core region within TES, named TESCO, is an important enhancer for mammalian sex determination. We show that TESCO of the genus Tokudaia lost enhancer activity caused by mutations in its SRY and SF1 binding sites. Two species of Tokudaia do not have the Y chromosome or SRY, and one species has multiple SRYs located on the neo-Y chromosome consisting of the Y fused with an autosome. The sequence of Tokudaia TESCO exhibited more than 83% identity with mouse TESCO, however, nucleotide substitution(s) were found in two out of three SRY binding sites and in five out of six SF1 binding sites. TESCO of all species showed low enhancer activity in cells co-transfected with SRY and SF1, and SOX9 and SF1 in reporter gene assays. Mutated TESCO, in which nucleotide substitutions found in SRY and SF1 binding sites were replaced with mouse sequence, recovered the activity. Furthermore, SRYs of the SRY-positive species could not activate the mutated TESCO or mouse TESCO, suggesting that SRYs lost function as a sex-determining gene any more. Our results indicate that the SRY dependent sex-determining mechanism was lost in a common ancestor of the genus Tokudaia caused by nucleotide substitutions in SRY and SF1 binding sites after emergence of a new sex-determining gene. We present the first evidence for an intermediate stage of the switchover from SRY to a new sex-determining gene in the evolution of mammalian sex-determining mechanism.