Schematic of wild-type UPF3B and UPF3B from affected individuals. (a) There are two recognized domains within the UPF3B protein: one at residues 48–150 (light gray), which is involved in binding to UPF2 (ref. 30), and the other, spanning residues 425–435 (white dots), through which UPF3B interacts with the components of the exon junction complex and Y14 in particular18. The three protein-truncating mutations (in families 1, 2 and 3) and one missense mutation (in family 4) of UPF3B, and the resulting proteins, are also shown. The position of the alternatively spliced exon 8 (residues 270–282) is indicated as a small white rectangle. (b) ClustalW multiple protein alignment of partial UPF3A and UPF3B orthologs. The amino acid residues that differ from the sequence of the human UPF3B are shaded. The highly conserved tyrosine (Y) at position 160 is indicated with an arrow and outlined with a box.

Protein blot analysis of UPF3B. Protein lysates from two control LCLs and LCLs from four affected individuals are shown. Cellular proteins were separated on a 8% acrylamide gel and transferred onto nitrocellulose membrane. The membrane was probed with the UPF3B_901 (peptide 1) primary polyclonal sheep antibody and subsequently probed with horseradish peroxidase (HRP)-conjugated secondary donkey anti-sheep. The signal was detected using the enhanced chemiluminescence (ECL) method (upper panel). The position of the full-length, ~58-kDa UPF3B protein is indicated by an arrow. The UPF3B protein was detected in protein lysates from controls but not from any of the four affected individuals tested. The additional, larger protein species detected by the hUPF3B_901 antibody in lysates LCLs from controls and affected individuals represents nonspecific binding of the donkey anti-sheep and/or immunoglobulin cross-reactivity. A control protein blot using the monoclonal mouse antibody SC-32233 (Santa Cruz Biotechnology) directed to GAPDH is also shown (lower panel). The same result was also achieved with the hUPF3B_913 antibody (specific to peptide 2; data not shown).

Facial features of the probands from families 1, 2, 3 and 4. (a) Individual III-1 from family 1, showing a long, thin face, broad forehead and maxillary hypoplasia. (b) Individual III-4 from family 2, showing a long, thin face. (c) Individual IV-1 from family 3, showing a long, thin face, prominent forehead, facial asymmetry, high nasal bridge and a prominent chin. (d) Individual II-5 from family 4, demonstrating elongated but otherwise normal facial appearance. We obtained informed consent to publish photos of affected individuals from all four families.

UPF3B mutations identified in this study. cDNA (NM_080632) and protein (NP_542199) annotation of individual mutations is shown for each family. (a) Family 1 (previously reported)29. A four-nucleotide deletion, 674_677delGAAA, has been identified in this family; it was present in the two affected brothers and their mother (II-2; 100% X-chromosome inactivation skewing) but was absent in the grandmother (I-2; random, 61:39 skewing), indicating a probable de novo mutation event in the mother (II-2). (b) Family 2 (ascertained in Australia). The mutation was present in both affected boys and the mother. (c) Family 3, from the UK. Three generations with affected males have been recorded. This mutation, which causes a PTC, was identified in four affected males (no sample was available from II-3) and three obligate female carrier mothers (II-1, III-1 and III-3) and was absent from three unaffected males (II-5, III-4 and IV-2). (d) Family 4 (two generations of affected males; from the USA). The X chromosome inactivation assay showed moderately to highly skewed inactivation in all carrier females in this family, whereas noncarriers showed random skewing (data not shown; see also Methods). Open symbols represent normal individuals, and filled squares represent affected males. Probands in each family are indicated with arrows. Individual generations are numbered with Roman numerals (I, II, III). WT, wild-type allele; MUT, mutant allele; NT, not tested. DNA sequence chromatograms of individual mutations and wild-type alleles are given in Supplementary Figure 1 online.

Analysis of RNA expression of the UPF3B and other genes in controls and affected individuals. (a) Mean expression (± s.d.) of UPF3B, UPF3A and UPF2 in affected individuals (n = 3, black box) and controls (n = 4, gray box), as determined by real-time PCR. Expression was measured in three independent real-time PCRs and was normalized against expression of the ACTB gene in the same sample using the relative standard curve method. * P = 0.01, Student's t test. (b) UPF3B mRNA expression is significantly downregulated in affected individuals (n = 3) with UPF3B PTCs compared with unaffected controls (n = 3), as shown by real-time qRT-PCR. Inhibition of translation by treatment of LCLs with 100 mg ml⁻¹ cycloheximide for 6 h released the UPF3B mRNA from NMD. Although the expression of UPF3B also increased in controls (n = 3) as a consequence of cycloheximide treatment, this increase was not as marked as in affected individuals. The differential increase of UPF3B expression in affected individuals versus controls as a consequence of cycloheximide treatment was statistically significant (P = 0.008) at the 5% significance level (analysis of variance (ANOVA)). Measurements of expression were normalized against the expression of the ACTB gene in the same sample. Gray boxes indicate untreated LCLs; black boxes indicate cycloheximide-treated LCLs. All samples were run in triplicate. Bars indicate s.d. (c) Mean expression (± s.d.) of SMG5, PANK2 and GADD45B mRNA in affected individuals (n = 3, black box) and controls (n = 4, gray box) by real-time PCR. Expression was measured in two independent real-time PCRs and was normalized against ACTB expression using the relative standard curve method. * P < 0.01 (Student's t test).

Tissue expression profile of human UPF3B and UPF3A mRNA isoforms. Selected RNAs from the Total Human RNA Master Panel II (Clontech) were subjected to reverse transcription. The efficiency of the reaction was tested by PCR using primers specific to the ubiquitously expressed ESD gene (bottom panel). The expression of the two isoforms of either UPF3B or UPF3A were assessed by semiquantitative RT-PCR across the alternatively spliced exon 8 of UPF3B with primers UPF3B Ex5F and UPF3B Ex9R (upper panel), and across exon 4 in UPF3A with primers UPF3A Ex1F and UPF3A Ex6n7R (middle panel). The alternative isoforms, labeled isoform 1 and isoform 2, and the resulting two heteroduplexes, migrating as one product above isoforms 1 and 2, are indicated with arrowheads.