An XRCC4 splice mutation associated with severe short stature, gonadal failure, and early-onset metabolic syndrome

Abstract

Context: Severe short stature can be caused by defects in numerous biological processes including defects in IGF-1 signaling, centromere function, cell cycle control, and DNA damage repair. Many syndromic causes of short stature are associated with medical comorbidities including hypogonadism and microcephaly.

Objective: To identify an underlying genetic etiology in two siblings with severe short stature and gonadal failure.

Design: Clinical phenotyping, genetic analysis, complemented by in vitro functional studies of the candidate gene.

Setting: An academic pediatric endocrinology clinic.

Patients or other participants: Two adult siblings (male patient [P1] and female patient 2 [P2]) presented with a history of severe postnatal growth failure (adult heights: P1, -6.8 SD score; P2, -4 SD score), microcephaly, primary gonadal failure, and early-onset metabolic syndrome in late adolescence. In addition, P2 developed a malignant gastrointestinal stromal tumor at age 28.

Intervention(s): Single nucleotide polymorphism microarray and exome sequencing.

Results: Combined microarray analysis and whole exome sequencing of the two affected siblings and one unaffected sister identified a homozygous variant in XRCC4 as the probable candidate variant. Sanger sequencing and mRNA studies revealed a splice variant resulting in an in-frame deletion of 23 amino acids. Primary fibroblasts (P1) showed a DNA damage repair defect.

Conclusions: In this study we have identified a novel pathogenic variant in XRCC4, a gene that plays a critical role in non-homologous end-joining DNA repair. This finding expands the spectrum of DNA damage repair syndromes to include XRCC4 deficiency causing severe postnatal growth failure, microcephaly, gonadal failure, metabolic syndrome, and possibly tumor predisposition.

Figure 1.

A and B, Height and weight curves from ages 2–20 in P1 (A, blue lines) and P2 (B, red lines). The corrected skeletal age, as determined by serial bone age assessments (Greulich and Pyle,) is depicted by the open squares. C, Photograph of P1 (middle), P2 (right), and one of the study authors (left, 176 cm) in 2009, in which both patients show severe short stature, microcephaly, and short necks.

Figure 2.

XRCC4 c.246T>G is a splicing mutation. XRCC4 genomic and cDNA from PBMCs and primary dermal fibroblasts were analyzed. A, Electropherogram confirmed homozygous c.246T>G (highlighted) in P1 and P2 and heterozygosity in carrier S1. Amino acid residues are indicated. B, In silico donor splice site predictions (http://www.fruitfly.org/seq_tools/splice.html). Schematic of exon 3-intron 3-exon 4, donor site score indicated, and sequences corresponding to cryptic 5′ donor site that is generated by C.246T>G, are shown. C, RT-PCR products of XRCC4 cDNA from fresh PBMCs. Coverage of fragments 1 and 2 shown in schematic of XRCC4 cDNA. UTR, untranslated region; ORF, open reading frame that encodes XRCC4 protein; N, normal. D, Sanger sequencing of XRCC4 cDNA. Nucleotide c.246T is highlighted.

Figure 3.

Patient fibroblasts demonstrate a defect in NHEJ. Top, Schematic demonstrating a DSB in the middle of a 6-bp repeat. This can be repaired either via NHEJ (left) or microhomology (right). When repaired via microhomology, a BstXI restriction site is introduced that will result in the generation of multiple shorter DNA bands on the gel. Bottom, MMEJ assay performed in fibroblasts from a normal control, a DNA ligase IV-deficient patient, and proband P1. P1 and the DNA ligase IV-deficient fibroblasts both demonstrate multiple smaller DNA bands after the addition of BstXI, indicating preferential use of the MMEJ pathway for DNA repair.