Genetic Analysis of Syndromic and Nonsyndromic Patients With Craniosynostosis Identifies Novel Mutations in the TWIST1 and EFNB1 Genes.

INTRODUCTION: Craniosynostosis, the premature fusion of cranial sutures, is usually divided into 2 major categories: syndromic and nonsyndromic. Mutations in the FGFR1, FGFR2, FGFR3, TWIST1, and EFNB1 genes cause the common craniosynostosis syndromes Muenke, Crouzon and Crouzon with acanthosis nigricans, Apert, Pfeiffer, Saethre-Chotzen, and Craniofrontonasal. Overlapping features among craniosynostosis syndromes, phenotypic heterogeneity even within the same syndrome, especially in the case of Muenke syndrome, and inadequate clinical evaluation can lead to misdiagnosis, which molecular testing can help clarify. OBJECTIVE: The aim of this study is to investigate the underlying genetic cause in 46 patients with syndromic or nonsyndromic craniosynostosis by direct sequencing and/or microdeletion/microduplication analysis of the FGFR1-3, TWIST1, and EFNB1 genes. RESULTS: Genetic analysis identified 3 novel mutations, c.413T>C - p.(Leu138Pro) [p.(L138P)] in TWIST1, the previously reported c.373G>A - p.(Glu125Lys) [p.(E125K)], and c.717dupA - p.(Leu240IlefsTer79) [p.(L240fs)] mutation in EFNB1 gene as well as 6 previously known mutations and a heterozygous TWIST1 gene deletion. The 2 novel mutations within EFNB1 gene arose de novo, but the novel mutation p.(L138P) within TWIST1 gene was inherited from the patient's father, who was found to be mosaic for the mutation. To our knowledge, this is the first case of mosaicism described for TWIST1 gene. CONCLUSIONS: The contribution of molecular genetic analysis to the diagnosis of patients with syndromic craniosynostosis was useful because some were originally misdiagnosed. Conversely, thorough clinical evaluation can guide molecular testing and result in a correct diagnosis.

A novel de novo mutation within EFNB1 gene in a young girl with craniofrontonasal syndrome.

Craniofrontonasal syndrome is mainly characterized by frontonasal dysplasia, telorbitism, a broad nasal root, and frequently a bifid nose and coronal craniosynostosis. Craniofrontonasal syndrome is an X-linked disorder with an unusual pattern of inheritance because heterozygous females are more severely affected than hemizygous males. The craniofrontonasal syndrome-causing gene is EFNB1, localized in the border region of chromosome Xq12 and Xq13.1, encoding for protein ephrin-B1. Here we aim to investigate the underlying genetic defect of a young girl with craniofrontonasal syndrome. The patient underwent surgical correction of her craniofacial deformities. Genetic analysis was carried out by polymerase chain reaction. Products of exon 2 of the EFNB1 gene were sequenced as well as digested with BpmI enzyme. A novel de novo missense mutation 373G>A was identified within the EFNB1 gene, leading to the replacement of glutamic acid at amino acid position 125 with lysine. The replacement of Glu125 with Lys, which lies within the G-H loop, part of the dimerization ligand-receptor interface, is expected to disrupt the interaction between the Eph receptor and ephrin B1 ligand, thus leading to craniofrontonasal syndrome.

FGFR3 related skeletal dysplasias diagnosed prenatally by ultrasonography and molecular analysis: presentation of 17 cases.

Fibroblast Growth Factor Receptor 3 (FGFR3) related skeletal dysplasias are caused by mutations in the FGFR3 gene that result in increased activation of the receptors causing alterations in the process of endochondral ossification in all long bones, and include achondroplasia, hypochondroplasia, thanatophoric dysplasia, and SADDAN. Reports of prenatal diagnosis of FGFR3 related skeletal dysplasias are not rare; however, the correlation between 2nd trimester ultrasonographic findings and underlying molecular defect in these cases is relatively poor. There is a need for specific ultrasound (U/S) predictors than can distinguish lethal from non-lethal cases and aid an earlier prenatal diagnosis. Here we present one familial and 16 sporadic cases with FGFR3 related skeletal dysplasia, and we evaluate biometric parameters and U/S findings consistent with the diagnosis of skeletal dysplasia. U/S scan performed even at the 18th week of gestation can indicate a decreased rate of development of the femora (femur length (FL) <5th centile), while the mean gestational age at diagnosis is still around the 26th week. The utility of other biometric parameters and ratios is discussed (foot length, BPD, HC, FL/foot, and FL/AC). Prenatal cytogenetic and molecular genetic analyses were performed. A final diagnosis was reached by molecular analysis. In two cases of discontinued pregnancy, fetal autopsy led to a phenotypic diagnosis and confirmed the prenatal prediction of lethality. We conclude that the combination of U/S and molecular genetic approach is helpful for establishing an accurate diagnosis of FGFR3-related skeletal dysplasias in utero and subsequently for appropriate genetic counselling and perinatal management.

Easy, rapid, and cost-effective methods for identifying carriers of recurrent GJB2 mutations causing nonsyndromic hearing impairment in the Greek population.

A variety of techniques have been developed for screening the GJB2 gene for known and unknown mutations, especially the most common mutation in the Caucasian population, the c.35delG. Other mutations that have been so far characterized in the GJB2 gene seem to have different geographical distributions, and therefore there is an interest in identifying recurrent mutations specific for each population and developing easy and rapid screening techniques. Here we present easy screening protocols for already identified recurrent mutations in the Greek population. Developing easy, rapid, and cost-effective screening methods will facilitate the detection of GJB2 recurrent mutation carriers, at large, in the Greek population.

Genetic skeletal disorders of the fetus and infant: pathologic and molecular findings in a series of 41 cases.

BACKGROUND: Genetic skeletal disorders of the fetus and infant are a large group of genetic disorders, comprising the groups formerly assigned as skeletal dysplasias (osteochondrodysplasias), dysostoses, and malformation syndromes with a skeletal component. Genetic skeletal disorders may be prenatally detected by ultrasonography or result in intrauterine or early postnatal death, constituting one difficult diagnostic field met by the pathologist who performs the perinatal autopsy. METHODS: In this retrospective study, we have gathered radiologic, physical, histopathologic, and molecular data regarding 41 cases of genetic skeletal disorders diagnosed among 1980 fetal and perinatal autopsies over a 10-year period. RESULTS: Our series of cases were classified according to the 2006 Nosology and Classification of Genetic Skeletal Disorders. The overall frequency of genetic skeletal disorders was 1:48 autopsies. The FGFR3 group and osteogenesis imperfecta type 2 were the more frequently encountered disorders. The mean gestational age at autopsy was 21.9 weeks (range, 12-37 weeks). A final diagnosis was obtained in 95% of cases. Genetic skeletal disorders were detected by prenatal ultrasound in 90% of cases, with a correct typing of the disorder achieved in only 34%. Molecular analysis was confirmative in 5 cases. CONCLUSIONS: The central role of the perinatal pathologist in collaboration with specialized services is essential for the correct interpretation of the radiologic, physical, and histopathologic findings, to accurately classify specific types of genetic skeletal disorders and enable genetic counseling.

Endothelial nitric oxide synthase gene polymorphisms in recurrent spontaneous abortions.

OBJECTIVE: The risk of miscarriage is enhanced by a variety of genetic and environmental factors. Previous studies indicated an association between endothelial nitric oxide synthase (eNOS) activity, and implantation and maintenance of pregnancy, but it is rather controversial whether polymorphisms of the gene encoding for eNOS are associated with recurrent spontaneous abortions (RSA). The aim of our study was to determine whether the 27 bp intron 4 repeat polymorphism (4VNTR) and a Glu298Asp missense mutation encoded by exon 7 of the eNOS gene are associated with an increased risk for recurrent spontaneous abortions (RSA), in the Greek population. METHODS: A total of 126 women who had at least three unexplained spontaneous abortions before 20 weeks of gestation, with the same partner, were included in the study group. The control group consistent of 130 women with at least two live childbirths and without history of abortions. All patients and controls were investigated for the two polymorphisms. To genotype the cohorts we used the PCR-RFLPs method. RESULTS: The observed frequencies of bb, ba, aa genotypes of the VNTR, in intron 4, polymorphism were 0.75, 0.24, 0.01, respectively, for the patient group and 0.73, 0.24, 0.03, respectively, for the control group. The observed frequencies of GG, GT, TT of the Glu298Asp polymorphism were 0.42, 0.45, 0.13, respectively, for the patient group and 0.47, 0.45, 0.08, respectively, for the control group. Statistical analysis of the results indicates no significant difference between the two groups, for both the two studied polymorphisms. CONCLUSION: Our results do not show any influence of the two polymorphisms, VNTR in intron 4 and Glu298Asp of the eNOS gene, on early pregnancy.

Prenatal diagnosis of achondroplasia presenting with multiple-suture synostosis: a novel association.

OBJECTIVE: We report an atypical case of a fetus presenting with a combined achondroplasia and multiple craniosynostosis phenotype. METHODS: Sonographic monitoring in conjunction with molecular genetic analysis was performed in a 32-gestational weeks fetus. RESULTS: Sonographic findings were consistent with a diagnosis of achondroplasia associated with multiple-suture synostosis. The most common G380R FGFR3 achondroplasia mutation was detected. CONCLUSION: The most common achondroplasia mutation should be considered for prenatal DNA testing in cases with ultrasound findings of achondroplasia and multiple-suture synostosis. This is crucial for the genetic counselling and perinatal management of the fetus.

Prevalence of GJB2 mutations in prelingual deafness in the Greek population.

OBJECTIVE: Mutations in the gene encoding the gap junction protein connexin 26 (GJB2) have been shown as a major contributor to prelingual, sensorineural, nonsyndromic, recessive deafness. One specific mutation, 35delG, has accounted for the majority of the mutations detected in the GJB2 gene in Caucasian populations. The aim of our study was to determine the prevalence and spectrum of GJB2 mutations in prelingual deafness in the Greek population. METHODS: In a collaboration with the major referral centers for childhood deafness in Greece, patients were examined by an extensive questionnaire to exclude syndromic forms and environmental causes of deafness and by allele-specific polymerase chain reaction (PCR) for the detection of the 35delG mutation. Patients heterozygous for the 35delG mutation were further analyzed by direct genomic sequencing of the coding region of the GJB2 gene. RESULTS: The 35delG mutation was found in 42.2% of the chromosomes in 45 familial cases of prelingual, nonsyndromic deafness (18 homozygotes and 2 heterozygotes) and in 30.6% of the chromosomes in 165 sporadic cases (45 homozygotes and 11 heterozygotes). Direct genomic sequencing in heterozygous patients revealed the L90P (2 alleles), W24X (2 alleles), R184P (2 alleles), and 291insA (1 allele) mutations. CONCLUSION: Mutations in the GJB2 gene are responsible for about one third of prelingual, sensorineural, nonsyndromic deafness in the Greek population, and allele-specific PCR is an easy screening method for the common 35delG mutation.