Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome.

Crouzon syndrome is an autosomal dominant condition causing premature fusion of the cranial sutures (craniosynostosis) and maps to chromosome 10q25-q26. We now present evidence that mutations in the fibroblast growth factor receptor 2 gene (FGFR2) cause Crouzon syndrome. We found SSCP variations in the B exon of FGFR2 in nine unrelated affected individuals as well as complete cosegregation between SSCP variation and disease in three unrelated multigenerational families. In four sporadic cases, the normal parents did not have SSCP variation. Finally, direct sequencing has revealed specific mutations in the B exon in all nine sporadic and familial cases, including replacement of a cysteine in an immunoglobulin-like domain in five patients.

Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes.

Mutations in the fibroblast growth factor receptor 2 (FGFR2) gene have been identified in Crouzon syndrome, an autosomal dominant condition causing premature fusion of the cranial sutures (craniosynostosis). A mutation in FGFR1 has been established in several families with Pfeiffer syndrome, where craniosynostosis is associated with specific digital abnormalities. We now report point mutations in FGFR2 in seven sporadic Pfeiffer syndrome patients. Six of the seven Pfeiffer syndrome patients share two missense mutations, which have also been reported in Crouzon syndrome. The Crouzon and Pfeiffer phenotypes usually breed true within families and the finding of identical mutations in unrelated individuals giving different phenotypes is a highly unexpected observation.

A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome.

Pfeiffer syndrome (PS) is one of the classic autosomal dominant craniosynostosis syndromes with craniofacial anomalies and characteristic broad thumbs and big toes. We have previously mapped one of the genes for PS to the centromeric region of chromosome 8 by linkage analysis. Here we present evidence that mutations in the fibroblast growth factor receptor-1 (FGFR1) gene, which maps to 8p, cause one form of familial Pfeiffer syndrome. A C to G transversion in exon 5, predicting a proline to arginine substitution in the putative extracellular domain, was identified in all affected members of five unrelated PS families but not in any unaffected individuals. FGFR1 therefore becomes the third fibroblast growth factor receptor to be associated with an autosomal dominant skeletal disorder.

Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome.

Apert syndrome is a distinctive human malformation comprising craniosynostosis and severe syndactyly of the hands and feet. We have identified specific missense substitutions involving adjacent amino acids (Ser252Trp and Pro253Arg) in the linker between the second and third extracellular immunoglobulin (Ig) domains of fibroblast growth factor receptor 2 (FGFR2) in all 40 unrelated cases of Apert syndrome studied. Crouzon syndrome, characterized by craniosynostosis but normal limbs, was previously shown to result from allelic mutations of the third Ig domain of FGFR2. The contrasting effects of these mutations provide a genetic resource for dissecting the complex effects of signal transduction through FGFRs in cranial and limb morphogenesis.

A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene.

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E. Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14-15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18. The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

Spectrum of craniosynostosis phenotypes associated with novel mutations at the fibroblast growth factor receptor 2 locus.

The causative relationship between several of the syndromic forms of craniosynostosis and mutations in the fibroblast growth factor receptor (FGFR) loci is now well established. However, within the group of patients with craniosynostosis, there are several families and sporadic cases whose clinical features differ in variable degrees from the classically described syndromes of craniosynostosis. In this communication we present novel FGFR2 mutations associated with a spectrum of craniosyostosis phenotypes in 4 sporadic cases and in one family in which craniosynostosis segregates. The mutation and phenotype data presented emphasise the clinical variability of mutations at this locus and underline the plasticity of the phenotype-genotype relationship in this important group of congenital malformation syndromes. Mutations found were tyrosine 105 to cysteine, glycine 338 to glutamic acid, serine 351 to cysteine and glycine 384 to arginine. These are the first reported mutations in the first immunoglobulin-like loop (tyrosine 105 to cysteine) and the transmembrane domain (glycine 384 to arginine) of FGFR2, providing further insights into the mechanism of abnormal receptor function in FGFR2 mutations.

A chromosomal breakpoint that separates the esterase D and retinoblastoma predisposition loci in a patient with del(13)(q14q31).

A patient with severe mental retardation and other congenital abnormalities who developed retinoblastoma was shown to have a deletion on the long arm of chromosome #13 with breakpoints in regions q14 and q31. Quantitation of enzyme activity of the esterase-D gene which, together with the retinoblastoma locus, is located in region 13q14 showed levels that were equal to those of normal controls. The 13q14 breakpoint, therefore, appears to have occurred between the two loci, which places the esterase D gene in a more proximal position in this band than the retinoblastoma locus.

Amitriptyline and weight gain: a biochemical and endocrinological study.

A study was carried out in 6 healthy volunteers to test the hypothesis that weight gain associated with amitriptyline treatment may be due to hypoglycaemia caused by increased circulating blood insulin. Subjects were treated with 50 mg amitriptyline b.d. for 28 days. Estimations made of serum levels of amitriptyline and its metabolite nortriptyline showed a steady state by the 10th day. No significant weight-gain was observed in any of the volunteers, although 2 reported an increase in appetite. There were no significant differences in any of the glucose tolerance curves, fasting or peak insulin levels or in the glucose/insulin curves for Days 0, 14 and 28.

Arginine 109 to glutamine mutation in a girl with ornithine carbamoyl transferase deficiency.

We studied DNA from 29 families with at least one member with ornithine carbamoyl transferase (OCT) deficiency and have found a mutation in the TaqI site within exon 5 of the OCT gene in a female presenting at the age of 21 months. Hybridisation with site specific oligonucleotides shows that the mutation is a C to T substitution resulting in a glutamine for arginine substitution at amino acid 109.

The need to screen all retinoblastoma patients for esterase D activity: detection of submicroscopic chromosome deletions.

Roughly 5% of all patients with retinoblastoma carry a constitutional chromosome deletion on the long arm of chromosome 13, which confers a prezygotic predisposition to tumour development. As offspring of deletion carriers have a 50% risk of inheriting the predisposition locus it is important to identify deletion carriers. The site of the esterase D gene to the often deleted region offers an objective means of deletion identification. The chromosomes of a patient with unilateral retinoblastoma, previously supposed to have a normal karyotype, were reexamined after the discovery that his red blood cells contained reduced activities of esterase D. A small sub-band deletion was found in chromosome region 13q14. These findings emphasise the importance of measurements of esterase D in all patients with retinoblastoma, even those with an apparently normal karyotype.

Deletion of chromosome region 13q14 is transmissible and does not always predispose to retinoblastoma.

During routine screening of retinoblastoma patients for esterase D activity in red blood cell lysates a patient was identified with only 50% of normal enzyme activity. Chromosome analysis showed that this patient had a small deletion within chromosome region 13q14. Parental studies showed that, whereas the father had normal enzyme levels, the mother had esterase D levels which were also 50% of normal and a similar small 13q14 deletion. Ophthalmological examination failed to demonstrate any retinal abnormality in either parent. Thus we present the first case not only of the direct transmission of a 13q14 deletion within a family but also of an individual in whom the deletion has not predisposed to tumour formation.

An assessment of the usefulness of electrophoretic variants of esterase-D in the antenatal diagnosis of retinoblastoma in the United Kingdom.

Fifty retinoblastoma families have been studied. In 41 it has been possible to determine the esterase-D phenotypes in all family members. Seven families were informative for the enzyme polymorphism and in all cases cosegregation of the retinoblastoma gene and esterase-D alleles was demonstrated, giving a lod score of 2.61. When combined with other published reports the cumulative lod score is 13.69 with no recombination in 45 meioses. In 10-15% of retinoblastoma families therefore, it is possible to offer prenatal diagnosis using the ESD protein polymorphism. The application of this test to the retinoblastoma population in the UK is limited by the low frequency of the rarer allele (0.116) and, as a result of genetic counseling, the smaller families generally associated with retinoblastoma.

The estimation of fetal haemoglobin in healthy adults by radioimmunoassay.

A radioimmunoassay (RIA) for the measurement of fetal haemoglobin (HbF) was established and used to determine the distribution of HbF in 2463 British adults between the ages of 18 and 65 years. Although a range in HbF values was similar in both genders a shift towards higher values is seen in females and the mean HbF% is higher, in females, at all ages between 18 and 55. A significant and different fall in HbF over the age range studied was noted between females (47%) and males (23%). The difference in mean HbF between the genders was less marked after age 40 years and a small rise in HbF production was measured in females between ages 50 and 54 years.

Effect of the esterase-D phenotype on its in vitro enzyme activity.

Esterase-D phenotypes and in vitro activity have been measured in red blood cells from 258 retinoblastoma patients and 73 unaffected relatives. Individuals with the 1-1 and 2-1 phenotypes showed distributions of enzyme activity which were not significantly different from each other. Individuals with the 2-2 phenotype, however, consistently showed a 25-30% lower level of enzyme activity. These results demonstrate the importance of determining the esterase-D phenotype in individuals with low ESD activity who might otherwise be assumed to carry a chromosome deletion at the esterase-D locus. We have also shown that, in vitro, the ESD enzyme is unstable over relatively short periods of time which, if uncontrolled, can give rise to a large variation in measured enzyme levels. The addition of b-mercaptoethanol to the assay buffer, which stabilises the enzyme, results in more consistent values being obtained within the same ESD phenotype. This feature could account in part for much of the variability in enzyme activity observed between different individuals in other studies.

Deletions of the esterase D locus from a survey of 200 retinoblastoma patients.

Esterase D levels from 200 retinoblastoma patients have been measured in an attempt to identify individuals carrying deletions of chromosome region 13q14. In this series 75% had bilateral tumours and 23% were familial. Of nine patients identified as having low esterase D levels, five had not previously been diagnosed as deletion carriers. These observations demonstrate the benefit of screening retinoblastoma populations for esterase D deficiency.

Genetic and cytogenetic analysis of patients showing reduced esterase-D levels and mental retardation from a survey of 500 individuals with retinoblastoma.

The authors have analysed the esterase-D levels in 500 retinoblastoma patients of whom 15 showed red cell enzyme activities of approximately 50% that of normal controls. Chromosome analysis of these 15 patients confirmed the presence of a deletion involving region 13q14 in all cases. Seven of the 15 cases had not previously been diagnosed and all of these showed sub-band deletions within 13q14. None of these seven patients were mentally retarded although the remaining eight who showed larger chromosome deletions demonstrated the full spectrum of psychomotor abnormalities associated with 13q deletions. Two other mentally retarded retinoblastoma patients with normal esterase-D activity showed no karyotypic abnormality, demonstrating that mental retardation cannot be taken to indicate a chromosome deletion in all cases. Eight of the 15 deletion cases were only unilaterally affected. The data presented in this article suggest that esterase-D quantitation could provide the primary means of detection of chromosome deletions in retinoblastoma patients.

Pelizaeus-Merzbacher disease: detection of mutations Thr181----Pro and Leu223----Pro in the proteolipid protein gene, and prenatal diagnosis.

A family with an apparent history of X-linked Pelizaeus-Merzbacher disease presented for genetic counseling, requesting carrier detection and prenatal diagnosis. RFLP analysis using the proteolipid protein (PLP) gene probe was uninformative in this family. A prenatal diagnosis on a chorionic villus sample (CVS) was carried out using single-strand conformation polymorphism (SSCP) analysis of a variant in exon 4 of the PLP gene. The fetus was predicted to be unaffected. Sequencing of the exon from the CVS, the predicted-carrier mother, and the obligate-carrier grandmother revealed an A-to-C change at nucleotide 541 in the two women but not in the fetus. As this change results in a Thr-to-Pro change at amino acid 181 in a region of the gene predicted to be part of a transmembrane segment, it was concluded that this was the mutation causing the disease in this family. In addition, in a second family, an exon 5 variant band pattern on SSCP analysis was shown by sequencing to be due to a T-to-C change at nucleotide 668. This results in a Leu-to-Pro change in a carrier mother and in her two affected sons. These results provide further examples of mutations in PLP that cause Pelizaeus-Merzbacher disease and illustrate the value of SSCP in genetic analysis.

Mutation analysis in CD40 ligand deficiency leading to X-linked hypogammaglobulinemia with hyper IgM syndrome.

Mutations in the gene encoding CD40 ligand have been shown to be the cause of X-linked hypogammaglobulinemia with hyper IgM (HIGM1). We have used the technique of single strand conformational polymorphism (SSCP) analysis to screen for mutations in this gene in affected boys from nineteen unrelated families. Sixteen novel mutations were identified in patients, comprising six patients with single base substitutions, two patients with single base insertions, six patients with deletions ranging from one to seven bases and two patients with large deletions at the 5' end of the gene. These mutations were distributed throughout the gene SSCP band shifts and/or alterations in restriction enzyme digestion sites could be used for unambiguous determination of carrier status in at-risk female relatives of most of the affected boys and, in some cases, prenatal diagnosis also can be offered.

Diurnal variation in the effects of insulin on blood glucose, plasma non-esterified fatty acids and growth hormone.

Insulin 0.05 mu/kg body weight was injected intravenously into 14 subjects both at 8 a.m. and 5 p.m. in random order 12 hrs after a 50 g glucose meal. Fasting glucose levels were similar in both cases but the 48percent plus or minus 10 percent fall in blood glucose in the morning was significantly greater (p smaller than 0.001) than that of 34 percent plus or minus 7 percent in the afternoon. Fasting plasma NEFA, however, varied markedly between 477 plus or minus 150 muEqlL in tth morning and 725 plus or minus 195 muEqlL in the afternoon (p smaller than 0.001) and the fall after insulin injection (64 percent plus or minus 14 percent) was greater in the afternoon than in the morning (47 percent plus or minus 15 percent) (p smaller than 0.001). There was an inverse relationship between proportional glucose disappearance and proportional NEFA disappearance (p smaller than 0.001). The calculated caloric change in plasma, the sum of the falls in glucose and NEFA, were very similar in both morning (2.2 plus or minus 0.5 Cals/1) and afternoon (2.3 plus or minus 0.5 Cals/1), i.e., in spite of the variations of glucose and NEFA metabolism produced by insulin at different times, the nett effect, in terms of energy, was the same. Plasma growth hormone response in the afternoon was found to be enhanced compared with the morning values, although the degree of hypoglycaemia was greater in the morning.