A case of Japanese cleidocranial dysplasia with a CBFA1 frameshift mutation

Cleidocranial dysplasia (CCD), which is caused by mutations of the core binding factor alpha 1 (CBFA1)/runt-related gene 2 (Runx2), is an autosomal, dominantly inherited disorder of high penetrance affecting skeletal ossification and tooth development. Recently, we found a novel frameshift mutation 383-T-insertion (S128F) in exon 3 in the CBFA1 gene of a Japanese classic CCD patient. We describe our detailed investigation of the patient with CCD associated with the CBFA1 mutation. The patient showed the characteristic expression of CCD, such as dysplasia of the clavicles, patent fontanelles, short stature, impacted supernumerary teeth, and delayed eruption of the permanent teeth. In addition to these characteristics, orthopantomography delayed ossification of the mandibular symphysis and a three-dimensional computed tomograph (3D-CT) analysis showed hypoplasia of the zygomatic arch. Furthermore, the acellular cementum of an impacted supernumerary tooth was absent in this patient. Thus, the CBFA1 mutation was critical for the pathogenesis of CCD in this patient.     

RUNX2 mutations in Taiwanese patients with cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant human skeletal disorder comprising hypoplastic clavicles, wide cranial sutures, supernumerary teeth, short stature, and other skeletal abnormalities. It is known that mutations in the human RUNX2 gene mapped at 6p21 are responsible for CCD. We analyzed the mutation patterns of the RUNX2 gene by direct sequencing in six Taiwanese index cases with typical CCD. One of the patients was a familial case and the others were sporadic cases. Sequencing identified four mutations. Three were caused by single nucleotide substitutions, which created a nonsense (p.R391X), two were missense mutations (p.R190W, p.R225Q), and the forth was a novel mutation (c.1119delC), a one-base deletion. Real time quantitative PCR adapted to determine copy numbers of the promoter, all exons and the 3'UTR region of the RUNX2 gene detected the deletion of a single allele in a sporadic case. The results extend the spectrum of RUNX2 mutations in CCD patients and indicate that complete deletions of the RUNX2 gene should be considered in those CCD patients lacking a point mutation detected by direct sequencing.     

PEBP2alphaA/CBFA1 mutations in Japanese cleidocranial dysplasia patients

Cleidocranial dysplasia (CCD) is an autosomal dominant human bone disease whose genetic locus has been located on chromosome 6p21, where the PEBP2alphaA/CBFA1 gene essential for osteogenesis also maps. Previously, several heterozygous mutations in PEBP2alphaA/CBFA1 were found in CCD patients. In this study, we identified six different types of mutations in PEBP2alphaA/CBFA1 in Japanese CCD patients. Four cases were similar to those reported previously: two were nonsense mutations in the Runt domain, one was a hemizygous deletion, and the other was a missense mutation in the Runt domain which abolished the DNA-binding activity of Runx2/PEBP2alphaA/CBFA1. The remaining two mutations were novel: one had a heterozygous gt-to-tt mutation at the splice donor site (gt) between the exon3-intron junction, which resulted in abnormal exon3 skipping, and the other had a mutation in exon7, which led to the introduction of a translational stop codon in the middle of the transactivation domain. Thus, defects in either the DNA-binding domain or transactivation domain of Runx2/PEBP2alphaA/CBFA1 can cause CCD. The results not only provide a strong genetic evidence that mutations involving in PEBP2alphaA/CBFA1 contribute to CCD, but also provide a useful tool to study how Runx2/PEBP2alphaA/CBFA1 plays its pivotal role during osteoblastic differentiation.     

A novel missense mutation of the CBFA1 gene in a family with cleidocranial dysplasia (CCD) and variable expressivity

The aim of this study was to analyze the CBFA1 gene in a phenotypically variable family with autosomal dominant cleidocranial dysplasia (CCD). Five members of a family with CCD were characterized clinically. X-rays and photographs of the two clinically affected family members were taken. The genotype of all five affected family members was determined with the use of single strand conformation polymorphism (SSCP) and direct sequencing. A point-mutation in exon 2 (R148G) was detected in a patient with the full-blown clinical phenotype. His son, demonstrating the same mutation, showed only the dental CCD characteristics. No mutation could be found in the three clinically healthy family members. To conclude, a missense mutation in the CBFA1 gene was detected in a family with variably expressed CCD syndrome. A detailed clinical examination is necessary to detect minimally affected gene mutation carriers.     

The phenotypic spectrum of GLI3 morphopathies includes autosomal dominant preaxial polydactyly type-IV and postaxial polydactyly type-A/B; No phenotype prediction from the position of GLI3 mutations.

Functional characterization of a gene often requires the discovery of the full spectrum of its associated phenotypes. Mutations in the human GLI3 gene have been identified in Greig cepalopolysyndactyly, Pallister-Hall syndrome (PHS), and postaxial polydactyly type-A (PAP-A). We studied the involvement of GLI3 in additional phenotypes of digital abnormalities in one family (UR003) with preaxial polydactyly type-IV (PPD-IV), three families (UR014, UR015, and UR016) with dominant PAP-A/B (with PPD-A and -B in the same family), and one family with PHS. Linkage analysis showed no recombination with GLI3-linked polymorphisms. Family UR003 had a 1-nt frameshift insertion, resulting in a truncated protein of 1,245 amino acids. A frameshift mutation due to a 1-nt deletion was found in family UR014, resulting in a truncated protein of 1,280 amino acids. Family UR015 had a nonsense mutation, R643X, and family UR016 had a missense mutation, G727R, in a highly conserved amino acid of domain 3. The patient with PHS had a nonsense mutation, E1147X. These results add two phenotypes to the phenotypic spectrum caused by GLI3 mutations: the combined PAP-A/B and PPD-IV. These mutations do not support the suggested association between the mutations in GLI3 and the resulting phenotypes. We propose that all phenotypes associated with GLI3 mutations be called "GLI3 morphopathies," since the phenotypic borders of the resulting syndromes are not well defined and there is no apparent genotype-phenotype correlation.     

Characterization of a germline mosaicism in families with Lowe syndrome, and identification of seven novel mutations in the OCRL1 gene.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by major abnormalities of eyes, nervous system, and kidneys. Mutations in the OCRL1 gene have been associated with the disease. OCRL1 encodes a phosphatidylinositol 4, 5-biphosphate (PtdIns[4,5]P2) 5-phosphatase. We have examined the OCRL1 gene in eight unrelated patients with OCRL and have found seven new mutations and one recurrent in-frame deletion. Among the new mutations, two nonsense mutations (R317X and E558X) and three other frameshift mutations caused premature termination of the protein. A missense mutation, R483G, was located in the highly conserved PtdIns(4,5)P2 5-phosphatase domain. Finally, one frameshift mutation, 2799delC, modifies the C-terminal part of OCRL1, with an extension of six amino acids. Altogether, 70% of missense mutations are located in exon 15, and 52% of all mutations cluster in exons 11-15. We also identified two new microsatellite markers for the OCRL1 locus, and we detected a germline mosaicism in one family. This observation has direct implications for genetic counseling of Lowe syndrome families.     

Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the ΔC1897 frameshift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency. Received: 20 June 1997 / Accepted: 26 August 1997     

Analysis of mannose 6-phosphate uncovering enzyme mutations associated with persistent stuttering

GlcNAc-1-phosphodiester-N-acetylglucosaminidase ("uncovering enzyme" (UCE); EC 3.1.4.45) is a Golgi enzyme that mediates the second step in the synthesis of the mannose 6-phosphate lysosomal targeting signal on acid hydrolases. Recently, three mutations (two missense and one deletion/frameshift) in the NAGPA gene that encodes UCE have been identified in individuals with persistent stuttering. We now demonstrate that each mutation leads to lower cellular UCE activity. The p.R328C mutation impairs folding in the endoplasmic reticulum, resulting in degradation of a significant portion by the proteasomal system. The p.H84Q mutation also impairs folding and, in addition, decreases the specific activity of the enzyme that folds sufficiently to traffic to the Golgi. The p.F513SfsX113 frameshift mutation adds 113 amino acids to the C terminus of the cytoplasmic tail of the protein, including a VWLL sequence that causes rapid degradation via the proteasomal system. These biochemical findings extend the genetic data implicating mutations in the NAGPA gene in the persistent stuttering phenotype.     

Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number.

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.     

A novel mutation of adenomatous polyposis coli (APC) gene results in the formation of supernumerary teeth

Supernumerary teeth are teeth that are present in addition to normal teeth. Although several hypotheses and some molecular signalling pathways explain the formation of supernumerary teeth, but their exact disease pathogenesis is unknown. To study the molecular mechanisms of supernumerary tooth‐related syndrome (Gardner syndrome), a deeper understanding of the aetiology of supernumerary teeth and the associated syndrome is needed, with the goal of inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary teeth and colonic polyp lesion biopsies revealed that these patients exhibited significant pathological characteristics. APC gene mutations were detected by PCR and direct sequencing. We revealed the pathological pathway involved in human supernumerary tooth development and the mouse tooth germ development expression profile by RNA sequencing (RNA‐seq). Sequencing analysis revealed that an APC gene mutation in exon 15, namely 4292‐4293‐Del GA, caused Gardner syndrome in this family. This mutation not only initiated the various manifestations typical of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this case. Furthermore, RNA‐seq analysis of human supernumerary teeth suggests that the APC gene is the key gene involved in the development of supernumerary teeth in humans. The mouse tooth germ development expression profile shows that the APC gene plays an important role in tooth germ development. We identified a new mutation in the APC gene that results in supernumerary teeth in association with Gardner syndrome. This information may shed light on the molecular pathogenesis of supernumerary teeth. Gene‐based diagnosis and gene therapy for supernumerary teeth may become available in the future, and our study provides a high‐resolution reference for treating other syndromes associated with supernumerary teeth.     

Germline and somatic mutations in hMSH6 and hMSH3 in gastrointestinal cancers of the microsatellite mutator phenotype

Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.     

A Tyrosinase missense mutation causes albinism in the Wistar rat

Tyrosinase serves as a key enzyme in the synthesis of melanin. In humans mutations in the TYR gene are associated with type 1 oculocutaneous albinism (OCA1) that leads to reduced or absent pigmentation of skin, hair and eye. Various mutations causing OCA in man, mouse, rabbit and cattle have been identified throughout the Tyrosinase gene including nonsense, missense, frameshift and splice site alterations. Here we report a missense substitution at codon R299H in exon 2 of the Tyr gene in the albino Wistar rat. As this very exchange has already been described in OCA patients, our findings reinforce the significance of this region for normal catalytic activity of tyrosinase protein.     

X-linked hypophosphatemia in Polish patients. 1. Mutations in the PHEX gene

We present twenty-nine PHEX gene mutations extending our previous work, giving it to a total of 37 different mutations identified in Polish patients with familial or sporadic X-linked hypophosphatemia. Deletions, insertions and nucleotide substitutions leading to frameshift (27%), stop codon (29%), splice site (24%), and missense mutations (20%) were found. The mutations are distributed along the gene; exons 3, 4, 11, 12, 14, 15, 17, 20 and 22 are regions with the most frequent mutation events. Four mutations, P534L, G579R, R549X and IVS15+1nt, recurred in three, four, two and three unrelated patients, respectively. They have also been detected in affected persons from other countries. Twenty-eight mutations are specific for Polish population and almost all of them are unique. Most of the identified mutations are expected to result in major changes in protein structure and/or function.     

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor β subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .