Waardenburg综合征Ⅱ型患者MITF基因突变分析

Waardenburg综合征(WS)是临床上常见的常染色体显性遗传性耳聋综合征, MITF基因突变与部分Waardenburg 综合征Ⅱ型(WS2)病例的发病有关。MITF属于碱性螺旋-环-螺旋亮氨酸拉链转录因子家族, 能调节酪氨酸酶基因, 参与黑色素细胞的分化。文章报道了1个携带MITF基因点突变的3代Waardenburg综合征Ⅱ型中国家系。先证者表现为先天性重度感音神经性聋、虹膜异色、面部雀斑; 其他家系成员除一名仅表现为先天性耳聋外, 均表现为颜面、上肢雀斑和/或早白发。患者可检测到c.639delA杂合突变, 该突变在MITF基因第7外显子上产生了终止密码子(p.I220X), 突变产生的截短的MITF蛋白没有DNA结合活性。该突变是WS2病例中第3个位于MITF第7外显子的突变, 尚未见报道。该突变与已报道的位于第7外显子其他两个突变仅相差1个碱基, 氨基酸改变十分相似, 但在表型上有显著差别, 提示遗传背景对WS临床表型有重要影响。     

Homozygous truncating PTPRF mutation causes athelia

Athelia is a very rare entity that is defined by the absence of the nipple–areola complex. It can affect either sex and is mostly part of syndromes including other congenital or ectodermal anomalies, such as limb-mammary syndrome, scalp–ear–nipple syndrome, or ectodermal dysplasias. Here, we report on three children from two branches of an extended consanguineous Israeli Arab family, a girl and two boys, who presented with a spectrum of nipple anomalies ranging from unilateral hypothelia to bilateral athelia but no other consistently associated anomalies except a characteristic eyebrow shape. Using homozygosity mapping after single nucleotide polymorphism (SNP) array genotyping and candidate gene sequencing we identified a homozygous frameshift mutation in PTPRF as the likely cause of nipple anomalies in this family. PTPRF encodes a receptor-type protein phosphatase that localizes to adherens junctions and may be involved in the regulation of epithelial cell–cell contacts, peptide growth factor signaling, and the canonical Wnt pathway. Together with previous reports on female mutant Ptprf mice, which have a lactation defect, and disruption of one allele of PTPRF by a balanced translocation in a woman with amastia, our results indicate a key role for PTPRF in the development of the nipple–areola region.     

A mouse model of Waardenburg syndrome type IV resulting from an ENU-induced mutation in endothelin 3

A line of mutant mice (114-CH19) exhibiting white spotting and preweaning lethality was identified during an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. The trait segregated as a semidominant bellyspot with reduced penetrance. Homozygous mutant mice showed preweaning lethality, and exhibited white spotting over the majority of the body surface, with pigmented patches remaining around the pinnae, eyes and tail. Linkage analysis localized 114-CH19 on mouse chromosome 2, suggesting endothelin 3 (Edn3) as a candidate gene. Sequence analysis of Edn3 identified a G > A transversion that encodes an arginine to histidine substitution (R96H). This mutation is predicted to disrupt furin-mediated proteolytic cleavage of pro-endothelin that is necessary to form biologically active EDN3. This mutation is novel among human and mouse EDN3 mutants, is the first reported EDN3 ENU mutant, and is the second reported EDN3 point mutation. This study demonstrates the power of using ENU mutagenesis screens to generate new animal models of human disease, and expands the spectrum of EDN3 mutant alleles.     

Novel mutations of PAX3, MITF, and SOX10 genes in Chinese patients with type I or type II Waardenburg syndrome

Waardenburg syndrome (WS) is a rare disorder characterized by distinctive facial features, pigment disturbances, and sensorineural deafness. There are four WS subtypes. WS1 is mostly caused by PAX3 mutations, while MITF, SNAI2, and SOX10 mutations are associated with WS2. More than 100 different disease-causing mutations have been reported in many ethnic groups, but the data from Chinese patients with WS remains poor. Herein we report 18 patients from 15 Chinese WS families, in which five cases were diagnosed as WS1 and the remaining as WS2. Clinical evaluation revealed intense phenotypic variability in Chinese WS patients. Heterochromia iridis and sensorineural hearing loss were the most frequent features (100% and 88.9%, respectively) of the two subtypes. Many brown freckles on normal skin could be a special subtype of cutaneous pigment disturbances in Chinese WS patients. PAX3, MITF, SNAI2, and SOX10 genes mutations were screened for in all the patients. A total of nine mutations in 11 families were identified and seven of them were novel. The SOX10 mutations in WS2 were first discovered in the Chinese population, with an estimated frequency similar to that of MITF mutations, implying SOX10 is an important pathogenic gene in Chinese WS2 cases and should be considered for first-step analysis in WS2, as well as MITF.     

Screening of MITF and SOX10 Regulatory Regions in Waardenburg Syndrome Type 2

Waardenburg syndrome (WS) is a rare auditory-pigmentary disorder that exhibits varying combinations of sensorineural hearing loss and pigmentation defects. Four subtypes are clinically defined based on the presence or absence of additional symptoms. WS type 2 (WS2) can result from mutations within the MITF or SOX10 genes; however, 70% of WS2 cases remain unexplained at the molecular level, suggesting that other genes might be involved and/or that mutations within the known genes escaped previous screenings. The recent identification of a deletion encompassing three of the SOX10 regulatory elements in a patient presenting with another WS subtype, WS4, defined by its association with Hirschsprung disease, led us to search for deletions and point mutations within the MITF and SOX10 regulatory elements in 28 yet unexplained WS2 cases. Two nucleotide variations were identified: one in close proximity to the MITF distal enhancer (MDE) and one within the U1 SOX10 enhancer. Functional analyses argued against a pathogenic effect of these variations, suggesting that mutations within regulatory elements of WS genes are not a major cause of this neurocristopathy.     

A mouse model of Waardenburg syndrome type IV resulting from an ENU‐induced mutation in endothelin 3

A line of mutant mice (114‐CH19) exhibiting white spotting and preweaning lethality was identified during an N‐ethyl‐N‐nitrosourea (ENU) mutagenesis screen. The trait segregated as a semidominant bellyspot with reduced penetrance. Homozygous mutant mice showed preweaning lethality, and exhibited white spotting over the majority of the body surface, with pigmented patches remaining around the pinnae, eyes and tail. Linkage analysis localized 114‐CH19 on mouse chromosome 2, suggesting endothelin 3 (Edn3) as a candidate gene. Sequence analysis of Edn3 identified a G > A transversion that encodes an arginine to histidine substitution (R96H). This mutation is predicted to disrupt furin‐mediated proteolytic cleavage of pro‐endothelin that is necessary to form biologically active EDN3. This mutation is novel among human and mouse EDN3 mutants, is the first reported EDN3 ENU mutant, and is the second reported EDN3 point mutation. This study demonstrates the power of using ENU mutagenesis screens to generate new animal models of human disease, and expands the spectrum of EDN3 mutant alleles.     

Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.     

Waardenburg syndrome type II in a Chinese pedigree caused by frameshift mutation in the SOX10 gene

Waardenburg syndrome (WS) is a congenital hereditary disease, attributed to the most common symptoms of sensorineural deafness and iris hypopigmentation. It is also known as the hearing-pigmentation deficient syndrome. Mutations on SOXl0 gene often lead to congenital deafness and has been shown to play an important role in the pathogenesis of WS. We investigated one family of five members, with four patients exhibiting the classic form of WS2, whose DNA samples were analyzed by the technique of Whole-exome sequencing (WES). From analysis of WES data, we found that both the mother and all three children in the family have a heterozygous mutation on the Sex Determining Region Y - Box 10 (SOX10) gene. The mutation was c.298_300delinsGG in exon 2 of SOX10 ({"type":"entrez-nucleotide","attrs":{"text":"NM_006941","term_id":"1732746331"}}NM_006941), which leads to a frameshift of nine nucleotides, hence the amino acids (p. S100Rfs*9) are altered and the protein translation may be terminated prematurely. Further flow cytometry confirmed significant down-regulation of SOX10 protein, which indicated the SOX10 gene mutation was responsible for the pathogenesis of WS2 patients. In addition, we speculated that some other mutated genes might be related to disease phenotype in this family, which might also participate in promoting the progression of WS2.     

Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks

The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis.     

A nonsense mutation in MSX1 causes Witkop syndrome

Witkop syndrome, also known as tooth and nail syndrome (TNS), is a rare autosomal dominant disorder. Affected individuals have nail dysplasia and several congenitally missing teeth. To identify the gene responsible for TNS, we used candidate-gene linkage analysis in a three-generation family affected by the disorder. We found linkage between TNS and polymorphic markers surrounding the MSX1 locus. Direct sequencing and restriction-enzyme analysis revealed that a heterozygous stop mutation in the homeodomain of MSX1 cosegregated with the phenotype. In addition, histological analysis of Msx1-knockout mice, combined with a finding of Msx1 expression in mesenchyme of developing nail beds, revealed that not only was tooth development disrupted in these mice, but nail development was affected as well. Nail plates in Msx1-null mice were defective and were thinner than those of their wild-type littermates. The resemblance between the tooth and nail phenotype in the human family and that of Msx1-knockout mice strongly supports the conclusions that a nonsense mutation in MSX1 causes TNS and that Msx1 is critical for both tooth and nail development.     

Crigler-Najjar syndrome type 2: Novel UGT1A1 mutation

Crigler-Najjar syndrome type 2 is a rare cause for persistent unconjugated hyperbilirubinemia, inherited in an autosomal recessive manner. Even though it is compatible with normal life span, in the absence of prompt suspicion and intensive management it can prove fatal not only in the neonatal period but also during adult life. Here, we describe a case with a novel homozygous UGT1A1 p.Pro176Leu mutation.