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

Abstract

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.

Figure 1

Sonographic features of thanatophoric dysplasia (TD) with a view of the thorax demonstrating short ribs (A), a view of the head in a fetus with TD II and a cloverleaf skull (B), a longitudinal view of the thorax and abdomen demonstrating the ‘champagne cork’ appearance due to the very small chest (C), a 3-D image of the femur demonstrating slight bowing (D), a 3-D view of the face (E), a sagittal view showing the frontal bossing (F) also demonstrating a very small chest and a view of a hand illustrating the short fingers (G)

Figure 2

Fetal size charts for femur length (a), head circumference (b) and abdominal circumference (c) against gestational age in fetuses with thanatophoric dysplasia. Note the curvature in the femur length chart as growth declines significantly from 20 weeks gestation. Comparison with normal fetuses is demonstrated by overlaying measurements from affected fetuses on charts of fetuses of normal size (d–f, respectively) (derived from Chitty et al.–17) and by plots of Z-scores showing the deviation from the normal range (g–i, respectively)

Figure 3

Comparison of femur length in fetuses with thanatophoric dysplasia (closed circle) or achondroplasia (open circle) with normal fetuses (derived from Chitty et al.18) showing the distinctive pattern for TD, with little increase in size from the mid second trimester and achondroplasia, which follows the normal femoral centiles until around 25 weeks' gestation when it then falls away from the normal centiles

Figure 4

Restriction digest of PCR products for two thanatophoric dysplasia (TD) mutations. (A) The c.742C>T type I TD mutation was detected using PCR followed by digestion with AfeI, BsiHKAI and DraIII. cffDNA from a woman carrying an unaffected fetus is digested with AfeI (lane 3) but remains uncut using BsiHKAI (lane 4) and DraIII (lane 5); conversely, in an affected fetus, the AfeI site is destroyed, leaving some of the cffDNA undigested (lane 8), and a BbsI site (lane 9) and a DraIII site (lane 10) are created. (B) The c.1948A>G mutation was detected using digestion with the BbsI enzyme and two different primer sets. In the presence of an unaffected fetus, all cffDNA is digested by BbsI (lanes 3 and 8), whereas with an affected fetus, the BbsI restriction site is destroyed, leaving some of the cffDNA uncut (lanes 5 and 10)