Homozygous microdeletion of exon 5 in ZNF277 in a girl with specific language impairment

Abstract

Specific language impairment (SLI), an unexpected failure to develop appropriate language skills despite adequate non-verbal intelligence, is a heterogeneous multifactorial disorder with a complex genetic basis. We identified a homozygous microdeletion of 21,379 bp in the ZNF277 gene (NM_021994.2), encompassing exon 5, in an individual with severe receptive and expressive language impairment. The microdeletion was not found in the proband's affected sister or her brother who had mild language impairment. However, it was inherited from both parents, each of whom carries a heterozygous microdeletion and has a history of language problems. The microdeletion falls within the AUTS1 locus, a region linked to autistic spectrum disorders (ASDs). Moreover, ZNF277 is adjacent to the DOCK4 and IMMP2L genes, which have been implicated in ASD. We screened for the presence of ZNF277 microdeletions in cohorts of children with SLI or ASD and panels of control subjects. ZNF277 microdeletions were at an increased allelic frequency in SLI probands (1.1%) compared with both ASD family members (0.3%) and independent controls (0.4%). We performed quantitative RT-PCR analyses of the expression of IMMP2L, DOCK4 and ZNF277 in individuals carrying either an IMMP2L_DOCK4 microdeletion or a ZNF277 microdeletion. Although ZNF277 microdeletions reduce the expression of ZNF277, they do not alter the levels of DOCK4 or IMMP2L transcripts. Conversely, IMMP2L_DOCK4 microdeletions do not affect the expression levels of ZNF277. We postulate that ZNF277 microdeletions may contribute to the risk of language impairments in a manner that is independent of the autism risk loci previously described in this region.

Figure 1

Molecular characterization of the ZNF277 microdeletion in the discovery pedigree. (a) On the left, the results from the qPCR, on the right the discovery pedigree, where black indicates diagnosis of SLI and grey indicates language problems. (b) On the right, the results of PCR across the microdeletion breakpoints: only the allele with the microdeletion can be amplified and visualized as a band of 466 bp in the parents and in the proband. On the left, amplification of exon 5 of ZNF277 indicates the presence of at least one allele without the microdeletion. In both gels, 1 kb Plus DNA ladder was loaded at the extremities. (c) Sequence electropherograms from the PCR products spanning the microdeletion in ZNF277. The rectangle indicates the genomic position of the microdeletion in ZNF277. The 2 bp (TC), common to both ends, are delimited by dotted lines and circled in the reference sequence. bp, base pair, PCR, polymerase chain reaction, qPCR, qPCR.

Figure 2

Pedigree of the SLIC families carrying the ZNF277 microdeletion. Black filling indicates full diagnosis of SLI.

Figure 3

IMMP2L, DOCK4 and ZNF277 transcription levels in individuals with ZNF277 microdeletion. ZNF277 transcription levels in individuals with IMMP2L_DOCK4 microdeletion. The graph shows the ratio of IMMP2L, DOCK4 and ZNF277 transcript levels, normalized using GUSB as a reference. (a) Schematic representation showing ZNF277, DOCK4 and IMMP2L loci with respect to chromosome 7. The bars underneath show the chromosomal position of the two types of microdeletions that were analyzed. (b) the ratio has been calculated as an average of five samples for each group of individuals, belonging to four ASD families: ‘not-del' indicates the group of individuals with two wild-type copies of ZNF277, ‘del' the group of individuals with the heterozygous ZNF277 microdeletion. Two fragments were tested for the ZNF277 transcript: exons 1–2 (which lies outside of the microdeletion) and exons 3–5 (which encompasses the microdeletion). The expression pattern for the fragments was decreased in both cases when compared with individuals without the CNV, whereas in exon 5 it was significantly lower (P=0.035). Bars indicate the standard errors. (c) The ratio of ZNF277 expression has been calculated for two individuals of a family with an IMMP2L_DOCK4 microdeletion: the father, who has two normal copies of IMMP2L and DOCK4, and the mother, who carries the IMMP2L_DOCK4 deletion.