Exon IIIa Research Articles

Apert syndrome with S252W FGFR2 mutation and characterization using Phenomizer: An Indian case report

Human genetic disease needs differential diagnosis to optimize clinical management, enable prenatal detection, and genetic counselling. The current methods of robust DNA sequencing also require next generation phenotyping to match with for better interpretation of genotypic and phenotypic heterogeneity commonly observed. We report use of human ontology based phenotypic characterization with Phenomizer that gives statistical score for possible diagnoses based on which, the gene mutation was studied.A case of craniosynostosis which refers to a group of syndromes characterized by a premature fusion of skull was studied. The phenotypic features viz, dental crowding and dental malocclusion, bulbous nose, downslanted palpebral fissures, radial deviation of thumb, syndactyly of fingers, macrocephaly, and oxycephaly were entered to query the web-based tool Phenomizer which indicated high probability of mutation in FGFR2 gene. The proband, a 13-year-old male born to non-consanguineous parents showed mutation on FGFR2 gene at c.755C>G indicative of Apert syndrome. Apert syndrome is one of the most severe craniosynostosis syndromes with two possible mutations in the exon IIIa of FGFR2 gene reported in majority of the cases. This case study shows the importance of Phenomizer and molecular genetic analysis in differential diagnosis of genetic diseases.

Apert and Crouzon syndromes-Cognitive development, brain abnormalities, and molecular aspects.

Apert and Crouzon are the most common craniosynostosis syndromes associated with mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. We conducted a study to examine the molecular biology, brain abnormalities, and cognitive development of individuals with these syndromes. A retrospective longitudinal review of 14 patients with Apert and Crouzon syndromes seen at the outpatient Craniofacial Surgery Hospital for Rehabilitation of Craniofacial Anomalies in Brazil from January 1999 through August 2010 was performed. Patients between 11 and 36 years of age (mean 18.29 ± 5.80), received cognitive evaluations, cerebral magnetic resonance imaging, and molecular DNA analyses. Eight patients with Apert syndrome (AS) had full scale intelligence quotients (FSIQs) that ranged from 47 to 108 (mean 76.9 ± 20.2), and structural brain abnormalities were identified in five of eight patients. Six patients presented with a gain-of-function mutation (p.Ser252Trp) in FGFR2 and FSIQs in those patients ranged from 47 to78 (mean 67.2 ± 10.7). One patient with a gain-of-function mutation (p.Pro253Arg) had a FSIQ of 108 and another patient with an atypical splice mutation (940-2A →G) had a FSIQ of 104. Six patients with Crouzon syndrome had with mutations in exons IIIa and IIIc of FGFR2 and their FSIQs ranged from 82 to 102 (mean 93.5 ± 6.7). These reveal that molecular aspects are another factor that can be considered in studies of global and cognitive development of patients with Apert and Crouzon syndrome (CS). © 2016 Wiley Periodicals, Inc.

Mutations in the FGFR2 gene in Mexican patients with Apert syndrome.

Apert syndrome (AS) is a frequent acrocephalosyndactyly, with autosomal dominant inheritance. AS has been associated with mutations in fibroblast growth factor receptor 2 (FGFR2), and approximately 99% of cases show 2 of the frequent mutations located in exon IIIa (Ser252Trp or Pro253Arg). The purpose of the present study was to describe the mutations in exon IIIa of FGFR2 in Mexican AS patients and the relationships with clinical features. Exon IIIa of FGFR2 from 6 AS patients was amplified by polymerase chain reaction. Mutations in exon IIIa of the FGFR2 gene were identified by digestion with the restriction endonuclease Bstx1 and polyacrylamide gel electrophoresis. PCR fragments were cloned into the PCR 2.1 vector, and both DNA strands were sequenced using the T7 promoter and M13 universal cloning region oligonucleotides. Sequence alignment was performed using the MEGA software version 5. The patients' major clinical features included craniosynostosis, hypertelorism, proptosis, otitis media, midfacial hypoplasia, rhizomelic shortening, and hyperhidrosis. Mutation S252W was present in 4 patients, while the other 2 patients had P253R. In conclusion, either S252W or P253R mutations were present independently in AS patients; however, the 2 mutations were not found together. None of the clinical features were associated with any of the mutations, suggesting that other mutations may be involved in the development of this syndrome.

Clinicogenetic Study of Turkish Patients With Syndromic Craniosynostosis and Literature Review

BackgroundFibroblast growth factor receptor 2 mutations have been associated with the craniosynostotic conditions of Apert, Crouzon, Pfeiffer, Saethre-Chotzen, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes in various ethnic groups. MethodsThirty-three unrelated Turkish patients (12 with Apert syndrome, 14 with Crouzon syndrome, six with Pfeiffer syndrome, and one with Saethre-Chotzen syndrome) and 67 nonsyndromic craniosynostosis patients were screened for mutations in exons IIIa and IIIc of the FGFR2 gene by denaturing high-performance liquid chromatography and confirmed by direct sequencing. ResultsWe detected several pathogenic mutations in 11/33 (33%) patients with Apert syndrome (four with p.Pro253Arg; seven with p.Ser252Trp) and 8/33 (24%) patients with Crouzon syndrome (three with p.Trp290Arg, one with p.Cys342Tyr, p.Cys278Phe, p.Gln289Pro, and a novel p.Tyr340Asn mutation) and five (15%) with Pfeiffer syndrome (p.Cys342Arg, p.Pro253Arg, p.Trp290Arg, and p.Ser351Cys). No FGFR2 gene mutation was detected in any of the patients with Saethre-Chotzen syndrome and nonsyndromic craniosynostosis. ConclusionsOur results indicate that the majority of Turkish patients with syndromic craniosynostosis have detectable genetic changes with an overall frequency of 72.7%. Because this is the first molecular genetic report from a Turkish cohort, the identified spectrum profile of FGFR2 mutations of the syndromic craniosynostotic patients would be very helpful for understanding the genotype–phenotype relationship and has a great value for diagnosis, prognosis, and genetic counseling.

Raised Intracranial Pressure Is Frequent in Untreated Nonsyndromic Unicoronal Synostosis and Does Not Correlate with Severity of Phenotypic Features

In a small number of children with unicoronal synostosis, the phenotype is mild and the aesthetic benefit of surgical correction is potentially outweighed by surgical risk. Raised intracranial pressure, however, would necessitate intervention. The authors documented the incidence of raised intracranial pressure in children with mild features and/or parental reluctance to proceed directly to surgery. A retrospective review of all children with (1) a clinical diagnosis of nonsyndromic unicoronal synostosis; (2) unicoronal synostosis confirmed on computed tomographic scanning; (3) negative family history for unicoronal synostosis; and (4) negative genetic screening (including P250R encoded by FGFR3, exons IIIa and IIIc in FGFR2 and TWIST1) was completed. Of the 80 children studied, seven (9 percent) underwent formal intracranial pressure monitoring because of a combination of mild clinical features (n = 4) and/or parental reluctance to proceed with surgery (n = 3). Intracranial pressure monitoring was reported as high in three (42 percent), borderline in two (29 percent), and normal in two patients (29 percent). The findings did not correlate with the degree of clinical deformation or age at presentation. There was little correlation with clinical and radiographic features of raised intracranial pressure in this genetically screened nonsyndromic group. High or borderline raised intracranial pressure was present in five of seven cases. The risk did not correlate with degree of deformity or age at presentation. Formal intracranial pressure monitoring should be considered in all children with unicoronal synostosis not proceeding directly to surgical intervention, in combination with routine long-term follow-up and repeated intracranial pressure monitoring where indicated. Risk, V.

Growth Hormone Deficiency in a Case of Crouzon Syndrome with Hydrocephalus

Crouzon syndrome is one of the most common craniofacial syndromes and is inherited as autosomal dominant with variable expression. We report an 11 and a half-year-old boy with Crouzon syndrome with severe growth retardation. He had hydrocephalus since infancy and recently suffered from frequent dizziness. His bone age was only 5 years according to the Greulich and Pyle atlas. Magnetic resonance imaging showed shallow orbits, obstructive hydrocephalus, and cerebellar tonsil herniation. Growth hormone provocative tests revealed a reduced peak growth hormone response in both insulin and clonidine tests. Severe iron deficiency anemia was noted at the same time. Molecular analysis identified a common mutation point of Cys278Phe for Crouzon syndrome in exon IIIa of the fibroblast growth factor receptor 2 (FGFR2) gene. Since growth retardation is not a common feature of Crouzon syndrome, we reviewed the literature for the incidence of hydrocephalus in Crouzon syndrome and the association with growth hormone deficiency.

Rare mutations ofFGFR2causing apert syndrome: identification of the first partial gene deletion, and anAluelement insertion from a new subfamily

Apert syndrome (AS) is a severe disorder, characterized by craniosynostosis and complex syndactyly of the hands and feet. Two heterozygous gain-of-function substitutions (Ser252Trp and Pro253Arg) in exon IIIa of fibroblast growth factor receptor 2 (FGFR2) are responsible for >98% of cases. Here we describe two novel mutations in FGFR2 in the two patients in whom a mutation had not previously been found in our cohort of 227 AS cases. The first is a 1.93-kb deletion, removing exon IIIc and substantial portions of the flanking introns. This is the first large FGFR2 deletion described in any individual with craniosynostosis. The other mutation is a 5' truncated Alu insertion into exon IIIc. This is the third Alu insertion identified in AS; all have occurred within an interval of only 104 bp, representing an enrichment of over a million-fold compared to the background genomic rate. We show that the inserted Alu element belongs to a small subfamily, not previously known to be mobile, which we term Alu Yk13. Both the deletion and insertion are likely to act by a similar gain-of-function mechanism in which disruption of exon IIIc leads to illegitimate mesenchymal expression of an FGFR2 spliceform containing the alternatively spliced exon IIIb. All the AS-associated Alu insertions have arisen in the paternal germline; we propose that their enrichment in FGFR2 is driven by positive selection of the mutant spermatogonial progenitors, a mechanism analogous to that explaining why the canonical AS nucleotide substitutions also reach exceptionally high levels in sperm.

Cloning the Genomic Sequence and Identification of Promoter Regions of Bovine Pyruvate Carboxylase

Pyruvate carboxylase (PC) catalyzes a pivotal reaction in gluconeogenesis and lipid metabolism in liver. In bovine the PC gene is expressed as six 5′ untranslated region (UTR) mRNA variants. The objectives for this study were to clone and sequence the bovine PC gene, determine the intron and exon organization and identify PC promoter region(s). Oligonucleotide sequences that corresponded to the 5′ UTR mRNA variants and coding sequence of bovine PC were used to isolate 2 clones from the RPCI-42 bovine bacterial artificial chromosome (BAC) library. Sequencing data confirmed the presence of regions for the 5′ UTR for bovine PC mRNA. The exon arrangement from 5′ to 3′ is 48 (exon I), 41 (exon II), 178 (exon IIIA and IIIB), and 185 (exon IV) bp. Three promoter regions, P3, P2, and P1, adjacent to exon I, II, and IIIA, respectively, were identified based on computer analysis of sequence data. Putative promoters were cloned into a firefly luciferase vector and transiently transfected into H4IIE rat hepatoma cells. All PC promoters demonstrated luciferase activity comparable with the minimal promoter luciferase vector and higher than the promoterless luciferase vector. In addition, PC promoter 1 exhibited greater luciferase activity compared with PC promoter 2 or 3. These data provide information about the arrangement of the 4 bovine PC 5′ UTR exons, the identity of the promoter regions for the bovine PC gene, and indicate differences in relative basal activity of the promoter regions.

Q289P Mutation in FGFR2 Gene Causes Saethre-Chotzen Syndrome: Some Considerations About Familial Heterogeneity

To describe the first report on a three-generation family presenting typical features of Saethre-Chotzen syndrome, in which the Q289P mutation in the FGFR2 gene was detected. Dysmorphological evaluation was performed by a clinical geneticist. Direct sequencing of the polymerase chain reaction-amplified coding region of TWIST and screening for the P250R mutation in the FGFR3 gene were performed. Exons IIIa and IIIc of FGFR2 were sequenced also. The mutation was confirmed by both restriction-enzyme digestion and allelic-specific polymerase chain reaction. Neither TWIST gene analysis nor analysis of the P250R mutation on gene FGFR3 showed mutation within the coding sequence. A nucleotide change from CAG to CCG in exon IIIa of the FGFR2 gene that caused a Q289P mutation was detected, although exon IIIc in the propositus was normal. These same results were detected in his mother, but no other members of the kindred presented clinical features consistent with Saethre-Chotzen syndrome. This mutation was previously reported in individuals with Crouzon and Jackson-Weiss syndromes. The FGFR2 mutation in the family with Saethre-Chotzen syndrome herein reported reinforces the idea of an interaction among TWIST and FGFR genes during development. Absence of the Q289P mutation in some affected individuals in this family is discussed.

Mutation analysis of Crouzon syndrome in Taiwanese patients

Crouzon syndrome is an autosomal-dominant disorder that causes premature fusion of the cranial suture. Crouzon, Pfeiffer, and Apert syndromes are caused by mutations in the extracellular, third immunoglobulin-like domain, and adjacent linker regions (exons IIIa and IIIc) of the fibroblast growth factor receptor 2 (FGFR2) gene. We screened 12 Crouzon syndrome patients for mutations in exons IIIa and IIIc of the FGFR2 gene by polymerase chain reaction (PCR) and direct sequencing. Mutations were detected in nine of 12 patients at amino acid positions 278, 281, 289, 342, and 354. More than half of the studied Crouzon patients carried a mutation resulting in either the loss or gain of a cysteine residue. A novel missense Ser354Phe substitution at exon IIIc of the human FGFR2 gene was found. According to our results, sequencing analysis of IgIII domain of the FGFR2 gene can lead to a genetic diagnosis of Crouzon syndrome.

Resolution of acne following therapy with an oral contraceptive in a patient with Apert syndrome

To the Editor: Apert syndrome is a genetic disorder characterized by irregular craniosynostosis, midfacial hypoplasia, syndactyly, and severe, difficult-to-control widespread acne, usually requiring isotretinoin treatment.1Jones K.L. Smith D.W. Smith's recognizable patterns of human malformation. W.B. Saunders Company, Philadelphia1996Google Scholar To our knowledge, our patient represents the first reported case of Apert syndrome whose severe acne resolved dramatically following therapy with an oral contraceptive. A female patient with Apert syndrome was first seen in the dermatology clinic at 15 years of age with a history of severe acne for 2 years, involving her face, chest, back, abdomen, and arms (Fig 1, A). She had tried various topical medications and oral erythromycin without good response. Oral isotretinoin was recommended as first line. However, given her family's concerns with the side effects, oral minocycline, and adapalene cream were started with mild improvement. At 17 years of age, she was started on a triphasic oral contraceptive (norgestimate/ethinyl estradiol) for a large ovarian cyst. Consequently, she saw dramatic improvement in her acne. The improvement was maintained as she continued on the oral contraceptive one year later with no adjunctive therapies (Fig 1, B). Apert syndrome is inherited in an autosomal dominant fashion; however, the vast majority of the cases are sporadic. The prevalence is approximately 15.5/1,000,000 births, and it affects both sexes in the same ratio. There is a significant association with advanced paternal age, and mutations occur on the paternal chromosome. Mutations in the fibroblast growth factor receptor 2 gene (FGFR2), which maps to chromosome 10q25-10q26, cause Apert syndrome. Specific missense substitutions involving adjacent amino acids (Ser252Trp and Pro253Arg) in the linker between the second and third extracellular immunoglobulin domains of the FGFR2 (exon IIIa) cause the majority of cases.1Jones K.L. Smith D.W. Smith's recognizable patterns of human malformation. W.B. Saunders Company, Philadelphia1996Google Scholar, 2Munro C.S. Wilkie A.O. Epidermal mosaicism producing localized acne: somatic mutation in FGFR2.Lancet. 1998; 352: 704-705Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar The dermatologic hallmark of Apert syndrome is severe inflammatory and comedonal acne, involving the face, chest, back, and also unusual areas such as the forearms, buttocks, and thighs.3Cohen M.M. Kreiborg S. Cutaneous manifestations of Apert syndrome.Am J Med Genet. 1995; 58: 94-96Crossref PubMed Scopus (44) Google Scholar FGFR2 is hypothesized to be involved in its pathogenesis.2Munro C.S. Wilkie A.O. Epidermal mosaicism producing localized acne: somatic mutation in FGFR2.Lancet. 1998; 352: 704-705Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar This acne is often resistant to conventional therapies. Five previous case reports have reported isotretinoin to be useful in controlling acne in Apert syndrome, though high doses and repeated or prolonged courses were required.4Parker T.L. Roth J.G. Esterly N.B. Isotretinoin for acne in Apert syndrome.Pediatr Dermatol. 1992; 9: 298-300Crossref PubMed Scopus (15) Google Scholar The efficacy of oral contraceptives to improve acne in healthy adolescents has been reported in many studies, including recent multicenter, randomized, placebo-controlled trials. Oral hormonal contraceptives affect acne in several ways. Estrogen improves acne by increasing levels of sex hormone-binding globulin (SHBG), thus decreasing free testosterone, and by suppressing gonadotropin secretion, thus reducing ovarian androgen production.5Lucky A.W. Henderson T.A. Olson W.H. Robisch D.M. Lebwohl M. Swinyer L.J. Effectiveness of norgestimate and ethinyl estradiol in treating moderate acne vulgaris.J Am Acad Dermatol. 1997; 37: 746-754Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar The progestin norgestimate used in this case has a low binding affinity for androgen receptors and SHBG. Patients with Apert syndrome are found to have an abnormal sensitivity to normal circulating levels of androgens. Thus, the antiandrogenic action of oral contraceptives would plausibly explain the underlying mechanism in controlling acne in Apert syndrome.

Alternative transcripts of rat slc19a1: Cloning, genomic organisation, tissue specific promoters and alternative splicing

Recently, the rat genome project revealed the genomic sequence of slc19a1, coding for the methotrexate carrier-1, identical to the reduced folate carrier-1 of humans, on rat chromosome 20. At the same time, we have cloned and analysed the complete or partial cDNAs of now at least six different transcripts from rat liver and kidneys. Alignment with the genomic sequence revealed seven exons. The first two non-coding exons, exon I and Ia were used alternatively in kidneys and liver, respectively, suggesting usage of alternative promoters. Three minor mRNA forms resulted from absent splicing of intron III, a shortened exon III (exon IIIa), and a shortened exon IV (exon IVa). The minor transcripts were predicted to result in translation products with 7 or 6 instead of 12 transmembrane domains (TMDs) and a peptide mass of 38, 39 and 40 kDa instead of 58 kDa.

Novel mutation in the tyrosine kinase domain of FGFR2 in a patient with Pfeiffer syndrome

Mutations in the fibroblast growth factor receptor 2 (FGFR2) cause a variety of craniosynostosis syndromes. The mutational spectrum tends to be narrow with the majority of mutations occurring in either exon IIIa or IIIc or in the intronic sequence preceding exon IIIc. Mutations outside of this hotspot are uncommon and the few identified mutations have demonstrated wide clinical variability, making it difficult to establish a clear-cut genotype-phenotype correlation. To better delineate the clinical picture associated with these unusual mutations, we describe a severely affected patient with Pfeiffer syndrome and a missense mutation in the tyrosine kinase (TK) domain of FGFR2.

Functional analysis of fibronectin isoforms in chondrogenesis: Full-length recombinant mesenchymal fibronectin reduces spreading and promotes condensation and chondrogenesis of limb mesenchymal cells

Fibronectin (FN), a large dimeric glycoprotein, functions primarily as a connecting molecule in the extracellular matrices of tissues by mediating both cell-matrix and matrix-matrix interactions. All members of the FN family are products of a single FN gene; heterogeneity arises from the alternative splicing of at least three regions (IIIB, IIIA, and V) during processing of a common primary transcript. During chick embryonic limb chondrogenesis, FN structure changes from B +A + in precartilage mesenchyme to B +A − in differentiated cartilage, and exon IIIA has been shown to be necessary for the process of mesenchymal cellular condensation, a requisite event that precedes overt expression of chondrocyte phenotype. This study aims to investigate the mechanistic action of the FN isoforms in mesenchymal chondrogenesis and, in particular, to identify the specific cellular function in mesenchymal condensation mediated by the mesenchymal (B +A +) FN isoform. Full-length cDNAs corresponding to four splice variants (B +A +, B +A −, B −A +, B −A −) of FN were constructed, and expressed the corresponding proteins using a baculovirus expression vector system. Cell adhesion assays with purified proteins showed that, although the relative levels of cell attachment were approximately the same, chick limb-bud mesenchymal cells spread up to 40% less on mesenchymal (B +A +) FN than on cartilage (B +A −) FN, (B −A +) FN, or plasma (B −A −) FN. Cellular condensation and chondrogenic differentiation were also promoted in high-density micromass cultures of limb mesenchymal cells plated onto B +A + FN. These observations suggest that the process of mesenchymal condensation is mediated at least in part by the enhanced ability of chondrogenic mesenchymal cells to migrate and aggregate as a consequence of residing in and interacting with mesenchymal FN. Our findings are consistent with and provide a mechanistic basis for previous observations that rounding of limb mesenchymal cells precedes the onset of chondrogenesis.

Three craniosynostotic patients with tracheal sleeve.

The gene was analysed in three craniosynostotic patients with a tracheal sleeve and other abnormalities. Although the majority of mutations are found in either exons IIIa or IIIc, this was not found in this study. It may be that patients with a tracheal sleeve are genetically heterogeneous.

Genetic and environmental risk factors for sagittal craniosynostosis.

The authors investigated whether genetic and environmental factors influence risk for sagittal craniosynostosis. Cases were ascertained from craniofacial clinics in the Baltimore-Washington metropolitan region. Controls were recruited from the Johns Hopkins newborn nursery and a large pediatric practice in Baltimore County. Forty-two probands with isolated, nonsyndromic sagittal craniosynostosis born in the mid-Atlantic region were included in this analysis. Controls are infants born in Maryland without any known birth defects (n = 182). Odds ratios (OR) and corresponding 95% confidence intervals (CI) were calculated. Cases were genotyped at several loci implicated in malformation syndromes including craniosynostosis. There were no elevated risks for craniosynostosis related to maternal or paternal smoking or maternal vitamin usage. Case mothers consumed less alcohol (OR = 0.38, 95% CI = 0.17-0.85) and had less education than control mothers ( < 0.001). All cases that were sequenced were negative for mutations at the following genes: exon IIIa 755C->G, (exons IIIa and IIIc,), exon IIIa, and exon 1. These findings suggest that whereas TWIST and the genes are important for syndromic craniosynostosis, they are unlikely to be involved in isolated sagittal craniosynostosis. Parental education and alcohol consumption were associated with sagittal craniosynostosis in this study.

Mutation analysis of Crouzon syndrome and identification of one novel mutation in Taiwanese patients.

Crouzon syndrome is an autosomal dominant disorder causing premature fusion of the cranial suture. Mutations have been reported in exon IIIa or IIIc of the fibroblast growth factor receptor 2 (FGFR2) gene. In the present study, nine unrelated Crouzon syndrome patients were screened for mutations in the two exons of FGFR2 by polymerase chain reaction and direct sequencing. Mutations were detected in 67% (6/9) of all cases. More than half the studied Crouzon patients carried a mutation resulting in either the loss or gain of a cysteine residue. A novel mutation, Tyr281Cys substitution, was discovered at exon IIIa. The mechanisms by which the same genotypes cause different phenotypes for each type of craniosynostosis syndrome in still uncertain. However, the molecular identification of the FGFR gene has made a great impact on the clinical classification of craniosynostosis syndromes; a new classification based on genotypes seems to be unavoidable.

Novel SNP at the common primer site of exon IIIa of FGFR2 gene causes error in molecular diagnosis of craniosynostosis syndrome.

Most mutations in Crouzon, Pfeiffer, and Apert syndromes are in the extracellular, third immunoglobulin-like domain and adjacent linker regions (exons IIIa and IIIc) of the fibroblast growth factor receptor 2 (FGFR2) gene. Using the published primers for PCR, a patient with Crouzon syndrome was found to be homozygous for a mutation that results in a Q289P amino acid substitution in FGFR2. Two additional patients; one with Apert syndrome and P253R mutation, the other with Pfeiffer syndrome and S267P mutation, also appeared to be homozygous. Using a new primer located 146 bp 5' of exon IIIa for PCR followed by sequencing revealed an A to G polymorphism at -62 [corrected] position of exon IIIa. All three patients were heterozygous for both the mutation and the polymorphism. These results indicate that the polymorphism and the mutation are not on the same chromosome. The single nucleotide polymorphism is located at the second to the last base of the 3' end of the published primer. This primer mismatch caused the failure of amplification of the normal chromosome and thus, the apparent homozygosity. The frequency of this novel polymorphism was determined to be 0.03 by studying 326 chromosomes from the general population. We propose that a new primer should be used for mutational analysis of exon IIIa of FGFR2 to avoid misdiagnosis caused by primer mismatch.

A Soluble Fibroblast Growth Factor Receptor is Released from HL-60 Promyelocytic Leukemia Cells: Implications for Paracrine Growth Control

The biological activities of fibroblast growth factors (FGF) are mediated by specific cell membrane receptors (FGFR), which have three immunoglobulin-like IgG domains in the extracellular region. The carboxy-terminal segment of the third IgG domain of FGFR1 could be encoded by different exons, designated IIIa, IIIb, or IIIc. While exons IIIb or IIIc encode receptor forms with both intracellular and extracellular domains, the FGF receptor becomes potentially a secreted form lacking the intracellular domain and the transmembrane region when exon IIIa is expressed. Using reverse transcription polymerase chain reaction, we have found that mRNAs encoding the nucleotide sequences of FGFR1-IIIa and FGFR1-IIIc are expressed in HL-60 cells. FGFR1-IIIa fragment was synthesized by a glutathione S-transferase gene fusion system. The purified 33kDa FGFR1-IIIa fragment fusion protein could bind [125I]-labelled FGF-2 in Western ligand blot analysis. Three species of proteins with the molecular weights of 82, 60, and 50 kDa were identified in serum-free, conditioned medium from HL-60 cells by Western blot using an antiserum against purified FGFR1-IIIa fragment fusion protein. Exposure to FGF-2 caused an increase in [3H]-thymidine incorporation into DNA of HL-60 cells and increased cell proliferation, but the addition of FGFR1-IIIa fragment fusion protein inhibited FGF-2-stimulated DNA synthesis and caused a dose-dependent inhibition of FGF-2-stimulated cell proliferation. The effects on DNA synthesis were partly reversed by antibody against the FGFR1-IIIa fragment. These results indicate that both cell membrane spanning and secreted FGF receptors are expressed in HL-60 cells, and that the actions of FGFs as paracrine growth factors could be modulated by secreted FGF receptor forms.

Rapid Detection of FGFR Mutations in Syndromic Craniosynostosis by Temporal Temperature Gradient Gel Electrophoresis

Several autosomal dominant craniosynostosis syndromes, including Crouzon, Pfeiffer, Apert, and Jackson-Weiss syndromes, are caused by mutations in the fibroblast growth factor receptor (FGFR) gene family (1). Mutations are not uniformly dispersed among these genes, and most mutations described to date are in exons IIIa and IIIc of the FGFR2 gene (2)(3). The advent of molecular analysis of these genes has allowed confirmatory testing in ambiguous cases and for those families desiring prenatal diagnosis. However, molecular analysis is difficult at present, especially for Crouzon syndrome, where current methods rely on the sequencing of the aforementioned exons. We report the rapid and inexpensive detection of FGFR mutations by a relatively novel screening assay, temporal temperature gradient gel electrophoresis (TTGE). TTGE is a heteroduplex detection method similar to denaturing gradient gel electrophoresis (DGGE). Like DGGE, but often unlike single-strand conformation polymorphism and heteroduplex analysis using mutation detection enhancement gels, TTGE is highly sensitive (4)(5)(6). However, TTGE is simpler than DGGE in avoiding the use of a chemical denaturing gradient gel and GC clamps (4). In TTGE, a homogeneous gel is bathed in a tank of buffer in which the temperature of the entire unit increases linearly throughout the electrophoretic run (5). After PCR, the products are heated and allowed to cool gradually, which leads to the formation of heteroduplex DNA if sequence heterogeneity is present. In dominant diseases, such as FGFR mutations that cause craniosynostosis syndromes, the sequence difference between the two alleles in the heteroduplex causes a physical bulge at the site of the sequence mismatch, …