Multiplex PCR in noninvasive prenatal diagnosis for FGFR3-related disorders.

Thanatophoric dysplasia and achondroplasia are allelic disorders caused by a constitutively active mutation in the FGFR3 gene. Because thanatophoric dysplasia is a lethal disorder and achondroplasia is non-lethal, they need to be distinguished after ultrasound identification of fetal growth retardation with short limbs. Accordingly, we have developed a noninvasive prenatal test using cell-free fetal DNA in the maternal circulation to distinguish thanatophoric dysplasia and achondroplasia. A multiplex PCR system encompassing five mutation hotspots in the FGFR3 gene allowed us to efficiently identify the responsible mutation in cell-free DNA in all examined pregnancies with a suspected thanatophoric dysplasia or achondroplasia fetus. This system will be helpful in the differential diagnosis of thanatophoric dysplasia and achondroplasia in early gestation and in couples concerned about the recurrence of thanatophoric dysplasia due to germinal mosaicism.

C-type natriuretic peptide plasma levels are elevated in subjects with achondroplasia, hypochondroplasia, and thanatophoric dysplasia.

CONTEXT: C-type natriuretic peptide (CNP) is a crucial regulator of endochondral bone growth. In a previous report of a child with acromesomelic dysplasia, Maroteaux type (AMDM), caused by loss-of-function of the CNP receptor (natriuretic peptide receptor-B [NPR-B]), plasma levels of CNP were elevated. In vitro studies have shown that activation of the MAPK kinase (MEK)/ERK MAPK pathway causes functional inhibition of NPR-B. Achondroplasia, hypochondroplasia, and thanatophoric dysplasia are syndromes of short-limbed dwarfism caused by activating mutations of fibroblast growth factor receptor-3, which result in overactivation of the MEK/ERK MAPK pathway. OBJECTIVE: The purpose of this study was to determine whether these syndromes exhibit evidence of CNP resistance as reflected by increases in plasma CNP and its amino-terminal propeptide (NTproCNP). DESIGN: This was a prospective, observational study. SUBJECTS: Participants were 63 children and 20 adults with achondroplasia, 6 children with hypochondroplasia, 2 children with thanatophoric dysplasia, and 4 children and 1 adult with AMDM. RESULTS: Plasma levels of CNP and NTproCNP were higher in children with achondroplasia with CNP SD scores (SDSs) of 1.0 (0.3-1.4) (median [interquartile range]) and NTproCNP SDSs of 1.4 (0.4-1.8; P < .0005). NTproCNP levels correlated with height velocity. Levels were also elevated in adults with achondroplasia (CNP SDSs of 1.5 [0.7-2.1] and NTproCNP SDSs of 0.5 [0.1-1.0], P < .005). In children with hypochondroplasia, CNP SDSs were 1.3 (0.7-1.5) (P = .08) and NTproCNP SDSs were 1.9 (1.8-2.3) (P < .05). In children with AMDM, CNP SDSs were 1.6 (1.4-3.3) and NTproCNP SDSs were 4.2 (2.7-6.2) (P < .01). CONCLUSIONS: In these skeletal dysplasias, elevated plasma levels of proCNP products suggest the presence of tissue resistance to CNP.

Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA.

OBJECTIVE: To improve the prenatal diagnosis of thanatophoric dysplasia by defining the change in fetal size across gestation and the frequency of sonographic features, and developing non-invasive molecular genetic diagnosis based on cell-free fetal DNA (cffDNA) in maternal plasma. METHODS: Fetuses with a confirmed diagnosis of thanatophoric dysplasia were ascertained, records reviewed, sonographic features and measurements determined. Charts of fetal size were then constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies and those complicated by achondroplasia. Cases in this cohort referred to our Regional Genetics Laboratory for molecular diagnosis using cffDNA were identified and results reviewed. RESULTS: Forty-two cases were scanned in our units. Commonly reported sonographic features were very short and sometimes bowed femora, frontal bossing, cloverleaf skull, short fingers, a small chest and polyhydramnios. Limb shortening was obvious from as early as 13 weeks' gestation, with minimal growth after 20 weeks. Analysis of cffDNA in three of these pregnancies confirmed the presence of the c.742C>CT (p.Arg248Cys) or the c.1948A>AG (p.Lys650Glu) mutation in the fibroblast growth factor receptor 3 gene. CONCLUSION: These data should improve the accuracy of the sonographic diagnosis of thanatophoric dysplasia and have implications for reliable and safe targeted molecular confirmation using cffDNA.

Sonographic and molecular diagnosis of thanatophoric dysplasia type I at 18 weeks of gestation.

Thanatophoric dysplasia is the most common type of lethal skeletal dysplasia. It can usually be diagnosed with ultrasound, but differential diagnosis with other osteochondrodysplasias is not always possible. Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have been demonstrated to cause two distinct subtypes of the disorder. We describe a case of thanatophoric dysplasia type I diagnosed at 18 weeks of gestation by ultrasonography. Genomic DNA obtained by chorionic villus sampling showed a C to G substitution at position 746 in the FGFR3 gene, resulting in a Ser249Cys substitution already known to be associated with type I disease. Implications for perinatal management are discussed.