PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features

Abstract

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function.

Figure 1

PUF60 gene diagram (above) indicating mutations found in our cohort which cluster in the second half of the coding region, apart from the single variant in exon 1. Note recurrent splicing variant in exon 8. Below is pictured the known protein structure and location of RRM domains in relation to the missense variants.

Figure 2

A de novo variant in PUF60 results in aberrant splicing at exon 8 splice-acceptor site. (a) The heterozygous, de novo PUF60 variant, c.604−2A>C was identified in patient II:1 through trio-based exome sequencing of family 3781. (b) Sequencing of skin-derived cDNA from patient II:1 (family 3781) showed normal splicing of exons 7 and 8 from one allele (top) and aberrant splicing with inclusion of the complete intron 7 from the mutant allele (bottom). Black arrows show the position of the oligonucleotide primers used for cDNA amplification and sequencing. The genomic context of the PUF60 gene is shown, with exons indicated as black boxes. The location of the PUF60 c.604−2A>C variant in intron 7 is indicated by a dotted red arrow, with the Sanger sequence trace from patient II:1 (family 3781) presented underneath. Variant nomenclature, exon numbering and the PUF60 messenger RNA sequence are based on sequence accession numbers NM_078480.2 (mRNA) and NPs_510965.1 (protein) and GenBank accession number NG_033879.1.

Figure 3

Spinal features (above): AP x-ray of cervical spine of patient 7 demonstrating hemivertebrae (left) and 3D-CT reconstruction of cervical spine of patient 3 demonstrating abnormalities of articulation of atlas with C1, vertebral bodies of C2/C3 and fusion of bodies of C6/C7/T1 and ocular features (below): right iris coloboma in patient 5.

Figure 4

Faces of patients (1, 2, 3, 4,6, 7, 8, 9, 10 and 11) with PUF60 variants, illustrating the thin upper lip, long philtrum, micrognathia and the flaring of eyebrows and narrow almond-shaped palpebral fissures, which are variably present. Patients 6 and 8 were assessed for CdLS and patients 7 and 10 had CREBBP analysis because of facial similarities with Rubinstein–Taybi syndrome. Patient 9 was investigated for Coffin–Siris syndrome.