We investigated a family of southern European origin with two offspring affected with SCD. Consanguinity was deemed unlikely, as the parents originated from geographically separated populations in Northern and Southern Italy. The first affected individual (II.1) was born at 41 weeks gestation, with a birth weight of 3400
g (75th centile) and length of 44
cm (<3rd centile). Newborn radiographs showed multiple segmentation anomalies and rib fusions (). She had a short trunk and short stature, and during the first year, it was noted that her span was greater than her length. Apart from a short neck and loss of thoracic (hypokyphosis) and lumbar (hypolordotic) curves, she developed normally with normal intelligence. Clinical examination showed a short broad neck with no lateral movement, but with a small amount of rotation and flexion in the anterior/posterior plane. There was limited trunk movement throughout the spine, although there was a small degree of rotation and forward flexion of the trunk in the lumbar spine. During childhood, she developed scoliosis convex to the left, centred about T10, with a compensatory curve in the lumbar spine centred on L3. She has 11 pairs of ribs on both sides with lateral fusion of the right first and second and third and fourth ribs. On the left, the fifth and sixth ribs are fused posteriorly. An MRI at 21 years of age () showed multiple segmentation anomalies in the cervical spine with butterfly and hemivertebrae. There is a sagittal cleft throughout the body of C2, and a defect (spina bifida) of the anterior ring of C1, with absence of ossification of the posterior ring. The arch of C1 is fused to C2 anteriorly. Some spinous processes are fused. There is superior displacement of the odontoid process. The junction of the medulla and cervical spinal cord is angulated anteriorly. Magnetic resonance angiography showed a dominant right vertebral artery with hypoplasia of the left vertebral artery, which does not communicate with the basilar artery. At 23 years of age, her pulmonary function showed a forced vital capacity that was 53% of predicted and a forced expiratory volume of one second (FEV1), which was 52% of predicted. Functional residual capacity was decreased in proportion to total lung capacity, consistent with a moderate restrictive ventilatory defect. Following correction for alveolar volume, the diffusing capacity for carbon monoxide (DLco) was 88% of predicted, in the lower half of its reference range consistent with normal gas transfer.
Figure 1 Radiograph (a) and T2-weighted coronal MRI images in the vertebral plane of individual II.1 (b) and coronal MRI image of individual II.3 (c), showing severe vertebral segmentation anomalies throughout the vertebral column. (d) Coronal view of a magnetic (more ...)
The second affected individual (II.3) was born by normal delivery at 39 weeks gestation with a birth weight of 3160
g (50th centile) and length of 43
cm (<3rd centile). Newborn radiographs showed eight ribs on both sides, with costal fusions on the right and multiple hemivertebrae with apparent block fusion L2–L3. The upper segment to lower segment ratio was 1.55. Postnatal growth remained less than the third centile, with span greater than height. At 13 years 3 months of age, height was 140.7
cm (<3rd centile), span was 162
cm (75th centile) and the upper segment/lower segment ratio was 0.85. He developed a prominent pectus excavatum with stiffness in the trunk and neck, thoracic hypokyphosis and lumbar hypolordosis. An X-ray of the wrist and ankle at the age of 10 years showed no evidence of carpal or tarsal fusions. MRI at 15 years of age in the bone mode showed multiple segmentation anomalies in the cervical and thoracic region (). Magnetic resonance angiography demonstrated a hypoplastic left vertebral artery with absence of both posterior communicating arteries ().
Genomic DNA from individual II.3 () was sequenced for the entire coding region and splice sites of the four genes previously shown to cause SCD (DLL3
and HES73, 4, 5, 6
genes showed no sequence deviations from the reference sequence. However, two heterozygous missense mutations (c.172A>G in exon 3 and c.556G>T in exon 4) were detected in HES7
, resulting in substitution of valine for isoleucine (I58V) and tyrosine for aspartic acid (D186Y). Further sequencing analysis of the entire family revealed that the other affected individual (II.1) was also compound heterozygous for I58V and D186Y, whereas each parent carried only a single missense mutation (the father carrying the I58V allele and the mother carrying the D186Y allele; ). One unaffected sibling also carried the D186Y allele, and the other was wild type at both sequence positions. Neither base change created a restriction fragment length polymorphism; hence, to confirm the presence of a bona fide
sequence alteration, products from at least two independent PCR reactions were sequenced for each affected individual. To demonstrate that these base changes were not common polymorphisms unassociated with the SCD phenotype, 110 ethnically matched control subjects (220 chromosomes) were sequenced. No control chromosome contained a mutation at either of these positions, giving approximately 80% power to distinguish a normal sequence variant from a mutation.16
In addition, the underlying base substitutions were not present in the NCBI SNP database.
Figure 2 Detection of HES7 mutations c.172A>G and c.556G>T. (a) Pedigree of family affected with SCD, with affected status indicated by black shading. The genotypes of each individual are shown using the predicted amino-acid change. (b) Electropherograms (more ...)
To investigate the effects of these base changes on HES7 function, we used two previously described cell culture transcription repression assays.17
HES family proteins repress transcription through two distinct mechanisms. These proteins bind directly to DNA through an N-box (CACNAG), using a basic region immediately amino-terminal to the helix-loop-helix domain (which is involved in homo- and heterodimerization with other bHLH family members). Co-repressors are then recruited to the promoter through interaction with a WRPW carboxy-terminal motif. They can also form heterodimers with the bHLH protein E47, thus preventing it (and other bHLH proteins that normally heterodimerize with E47 such as MyoD) from binding to E-boxes (CANNTG) and activating transcription. As no full-length human HES7
cDNA was available, we created I58W and D186Y mutations in mouse Hes7
, given that their bHLH regions have the same amino-acid sequence () and the entire proteins are 92% identical. Expression of wild-type Hes7 represses transcription from a beta-actin reporter with upstream N-boxes (), and represses E47-dependent transcription from a beta-actin reporter with upstream E-boxes () in mouse muscle satellite C2C12 cells. When the D186Y mutant form of Hes7 was used in the N-box assay, transcription was repressed to a significantly lesser extent (P
<0.0001) than with wild-type Hes7. In contrast, the I58V mutation showed no significant difference in the degree of repression compared with wild-type Hes7. The observed reduction in the ability of the D186Y mutant to repress transcription in the N-box assay might have been due to a reduction in protein expression level, or a destabilization of the Hes7 protein. We therefore tested this by transiently transfecting C2C12 with HA-tagged versions of wild-type and mutant Hes7 proteins. To monitor the relative protein stabilities, cells were treated with cycloheximide, and protein extracts were obtained at 0, 20, 40 and 60
min intervals. Hes7 protein levels were determined by means of western blots with beta-tubulin used as a loading control (Supplementary Figure). These experiments showed that all three proteins were expressed at similar levels under these conditions, and neither of the two mutant proteins was cleared from the cell in a manner different from that of the wild-type protein. This suggests that the D186Y mutant has reduced activity in the N-box assay due to an intrinsic disruption of its repressive activity. Similarly, in the E-box assay, the D186Y mutant Hes7 repressed to a significantly lesser extent (P
<0.0001) than did wild-type Hes7, whereas the I58V mutant repressed transcription to the same degree as did wild-type Hes7. This suggests that the D186Y mutation in the carboxy-terminal region of Hes7 also impairs the ability of Hes7 to heterodimerize with E47. The I58V mutant protein is not functionally compromised in either of the repression assays. However, we noted that the SCD phenotype is only observed when both I58V and D186Y mutations are present in an individual, and this may reflect a reduced capacity of the I58V mutant protein to dimerize with D186Y Hes7. We therefore co-expressed both I58V and D186Y Hes7 mutants in the N-box repression assay, and compared the level of repression with that of cells co-expressing wild-type and D186Y Hes7; wild-type and I58V Hes7; or each Hes7 protein singly (). In this assay, I58V, in combination with either wild-type or D186Y Hes7, had a statistically insignificantly different repressive activity compared with wild-type Hes7, suggesting that the I58V Hes7 protein is able to dimerize with D186Y Hes7 equally efficiently as with wild-type Hes7. This experiment also shows that, when co-expressed with D186Y Hes7, an equal amount of either wild-type or I58V Hes7 is sufficient to rescue normal repressive activity in the N-box assay.