Dubowitz syndrome is a complex comprised of multiple, genetically distinct and phenotypically overlapping disorders

Abstract

Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings' history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ∼ 3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463-65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of ≤ 1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined.

Figure 1. Frontal view of Patient 1 at age 41 years.

He was originally diagnosed with Dubowitz syndrome (Opitz et al, 1973; Walters and Desposito, 1985). This is the first published photograph of this seminal patient. Exome sequencing identified compound heterozygote mutations in LIG4, confirming the formal diagnosis of LIG4 syndrome. Dysmorphic features include microcephaly (<3^rd centile), bilateral downslanted palpebral fissures, low anterior hairline, and a long tubular nose with prominent bulbar tip.

Figure 2. Profile view of Patient 1 at age 41 years.

Dysmorphic features include a low posterior hairline, small (<−2 standard deviation), low-set ears and a high nasal bridge.

Figure 3. Frontal view of Patient 3 at age 15 years.

He was originally diagnosed with Dubowitz syndrome and was determined to harbor a 3.89 Mb deletion on chromosome 17q24.2. Dysmorphic features include bilateral upslanted palpebral fissures with apparent telecanthus, broad nasal bridge and a bulbous nose.

Figure 4. Profile view of Patient 3 at age 15 years.

Dysmorphic features include low set ears.

Figure 5. 2–3 cutaneous syndactyly and appearance of cutis marmorata telangiectasia congenita in Patient 3.

Figure 6. Median telomere lengths in lymphocytes, as determined by flow cytometry with fluorescent in situ hybridization (flow FISH).

Patients 1 (two time points), 2 and 3 are plotted against population norms. yrs  =  years; MTL  =  median telomere length; kb  =  kilobase

Figure 7. Colony survival assay demonstrates cellular radiosensitivity in Patient 1.

Fibroblasts from Patient 1 were highly sensitive to increasing doses of ionizing radiation. Experiments were performed in triplicate with the average of three experiments shown. WT  =  wild type; A-T  =  ataxia-telangiectasia control.

Figure 8. Colony survival assay demonstrates cellular radiosensitivity in Patients 2 and 3.

Lymphoblastoid cell lines from Patients 2 and 3 were radiosensitive, as compared to controls. Experiments were performed in triplicate with the average of three experiments shown. WT  =  wild type; A-T  =  ataxia-telangiectasia control. Ligase IV  =  Ligase IV-deficient control.

Figure 9. Neutral comet assay compatible with a DNA double-strand break repair disorder.

(a). Tail moment of WT, A-T and patient cells at 0, 30 minutes and 5 hours post-irradiation. All cells show damage at 30 minutes post-irradiation. WT returns to near baseline levels after 5 hours, while A-T and patient cells show long comet tails after 5 hours, indicating significant levels of unrepaired DNA. (b). Ratio of unrepaired DNA at 5/0 hours. A-T as well as Patient 2 and 3 cells show statistically significantly lower levels of repair at 5 hours post-irradiation compared to wild type cells (* p<0.05). Experiments were performed in triplicate with the average of three experiments shown. WT  =  wild type; A-T  =  ataxia-telangiectasia control.

Figure 10. Sanger sequencing of LIG4 c.613delT variant in Patients 1 and 2.

Figure 11. Sanger sequencing of LIG4 c.2440C>T variant in Patients 1 and 2.

Figure 12. Western blot of whole cell lysates from Patient 1 fibroblasts and Patient 2 lymphblastoid cell lines.

Patients 1 and 2 show an absence of ligase IV protein. Lanes were loaded with 50 µg protein/lane. LCL  =  lymphoblastoid cell line;