Genomic analysis of primordial dwarfism reveals novel disease genes

Abstract

Primordial dwarfism (PD) is a disease in which severely impaired fetal growth persists throughout postnatal development and results in stunted adult size. The condition is highly heterogeneous clinically, but the use of certain phenotypic aspects such as head circumference and facial appearance has proven helpful in defining clinical subgroups. In this study, we present the results of clinical and genomic characterization of 16 new patients in whom a broad definition of PD was used (e.g., 3M syndrome was included). We report a novel PD syndrome with distinct facies in two unrelated patients, each with a different homozygous truncating mutation in CRIPT. Our analysis also reveals, in addition to mutations in known PD disease genes, the first instance of biallelic truncating BRCA2 mutation causing PD with normal bone marrow analysis. In addition, we have identified a novel locus for Seckel syndrome based on a consanguineous multiplex family and identified a homozygous truncating mutation in DNA2 as the likely cause. An additional novel PD disease candidate gene XRCC4 was identified by autozygome/exome analysis, and the knockout mouse phenotype is highly compatible with PD. Thus, we add a number of novel genes to the growing list of PD-linked genes, including one which we show to be linked to a novel PD syndrome with a distinct facial appearance. PD is extremely heterogeneous genetically and clinically, and genomic tools are often required to reach a molecular diagnosis.

Figure 1.

Identification of a novel PD syndrome linked to CRIPT. (A,B) Pedigrees and clinical photographs of PD_F4_IV:2 and PD_F5_IV:4, respectively. The index is indicated by a red arrow, and asterisks denote individuals whose DNA was available for analysis. Note the strikingly similar clinical appearance (high forehead, mild proptosis, anteverted nares, flat nasal bridge, hypoplastic digits and talipes). Please also note the mottled hypopigmentation in PD_F4_IV:2. (C) Diagram of CRIPT (numbers indicate the number of exons, red color represents the coding exons while blue is the 5′ and 3′ UTR, and triangles indicate the sites of the mutations). (Upper panel) Sequence chromatogram showing the homozygous frameshift insertion in PD_F4_IV:2 and the heterozygous frameshift deletion in the parent of PD_F5_IV:4 (mutations are denoted by red lines).

Figure 2.

Identification of a novel Seckel syndrome locus linked to Chr10. (A) Pedigree and clinical photographs of the PD_F6 family. The index is indicated by a red arrow, and asterisks denote individuals whose DNA was available for analysis. Please note that the degree of consanguinity between II:1 and II:2 was not determined by parents (blue triangle) but was inferred by the size of the autozygome to be second- or third-degree consanguinity. The clinical photographs of the two affected (III:1 and II:5) show the typical Seckel “bird-like” face. (B) HomozygosityMapper reveals only one ROH on chromosome 10 shared between the two affected (red arrow), and AutoSNPa highlights a corresponding ROH of 7.6 Mb (boxed in red) exclusively shared between PD_F6_II:5 and PD_F6_III:1.

Figure 3.

Identification of a novel DNA2 mutation that causes increased senescence and DNA damage in a family with Seckel syndrome. (A) Upper panel shows DNA sequence chromatogram with the novel 1-bp deletion variant identified in the two affected members of PD_F6, and a schematic of the three transcripts of DNA2 affected by the mutation. The site of mutation on the sequence chromatogram is denoted by a red bar, and blue triangles denote the position of the variant on the three DNA2 transcripts. (B) Upper panel shows the gel image of the RT-PCR that reveals the presence of an aberrant band in the two patients as well as the reduction of the normal band in Pt1 and Pt2 (23% and 53%, respectively). Note the absence of the aberrant band in the two controls. Lower panel shows a cartoon of DNA2 mRNA (only exons 19–21 are shown) and the result of cloning experiments on the RT-PCR product obtained from the patient RNA which revealed the presence of five aberrant transcripts, all sharing the same abnormal donor site, while each had a different acceptor site. (C) Immunoblotting using antibody against DNA2. The protein level of DNA2 from cells derived from index individual (Pt) were 50% reduced in comparison to the normal type control (Ct), respectively (the average of two independent experiments was 60%). (D) Images of beta-gal-stained control cells (A) and patient (B) showing the presence of blue staining and flattening of patient cells (red triangle). Please note that patient and control fibroblasts had a comparable number of passages (6–7). (E) Representative images and the quantification graph in the assessment of “comet tail” lengths from patient cells transfected with shuttle (A) and DNA2 (B). There was a significant reduction in the number of cells with comet tails as well as the size of comet tails (P = 4.4 × 10⁻⁶) obtained from the patient cells after exogenous expression of DNA2. Scale bar = 50 μm.

Figure 4.

(A–C) Pedigree and stacked Venn diagrams illustrating the exome filtration scheme, sequence chromatogram, and the multisequence alignment of orthologs of the mutation identified in XRCC4. (D) Representative images and the quantification graph in the assessment of “comet tail” lengths from fibroblast cells treated with either XRCC4 or scrambled siRNA 4 h post-IR (5 Gy), showing that XRCC4 deficiency results in impaired DNA damage repair (P = 1.18 × 10⁻¹⁰).

Figure 5.

Pedigree, clinical photographs, and stacked Venn diagrams illustrating the exome filtration scheme and sequence chromatogram of the mutation identified in BRCA2 PD_F7. The index is indicated as a red arrow, and asterisks denote individuals whose DNA was available for analysis.