Kilquist syndrome: A novel syndromic hearing loss disorder caused by homozygous deletion of SLC12A2

Abstract

Syndromic sensorineural hearing loss is multigenic and associated with malformations of the ear and other organ systems. Herein we describe a child admitted to the NIH Undiagnosed Diseases Program with global developmental delay, sensorineural hearing loss, gastrointestinal abnormalities, and absent salivation. Next-generation sequencing revealed a uniparental isodisomy in chromosome 5, and a 22 kb homozygous deletion in SLC12A2, which encodes for sodium, potassium, and chloride transporter in the basolateral membrane of secretory epithelia. Functional studies using patient-derived fibroblasts showed truncated SLC12A2 transcripts and markedly reduced protein abundance when compared with control. Loss of Slc12a2 in mice has been shown to lead to deafness, abnormal neuronal growth and migration, severe gastrointestinal abnormalities, and absent salivation. Together with the described phenotype of the Slc12a2-knockout mouse model, our results suggest that the absence of functional SLC12A2 causes a new genetic syndrome and is crucial for the development of auditory, neurologic, and gastrointestinal tissues.

Keywords: NKCC1; absent salivation; cystic fibrosis; gut malrotation; uniparental isodisomy.

Figure 1.. Clinical phenotype.

A. A photo of the proband depicting an oblong face with a long lower-third face, forward placed midface, and bitemporal and bicoronal narrowing. B. Side profile of the proband showing long lower-third face, forward placed midface, and low-set ear with curling of the pinna C. Typical posture and positioning of proband. Although hypotonic, he has significant contractures. Scar visible from osteotomy. D. Computed tomography of the temporal bone showing vestibular dysplasia (left) as compared with an unaffected 5-month old. E. Magnetic resonance imaging of the brain, with axial section, showing decreased cerebral volume and enlarged arachnoid spaces (left) as compared with an unaffected 9-month old.

Figure 2.. Molecular analysis of SLC12A2 in proband.

A. Upper, schematic representation of wild type SLC12A2 locus. Middle, structure of proband’s SLC12A2 locus showing homozygous deletion of 22kb and inversion of 34 nucleotides. Arrows indicate position of forward and reverse primers used for PCR amplification. Lower, agarose gel image showing amplification of genomic DNA demonstrates homozygous deletion in proband and heterozygous deletion in his father. The proband’s genomic DNA from blood amplifies only when primers are used outside of the deletion. The father’s DNA amplifies in all cases, showing he is a heterozygote. The mother’s genomic DNA does not amplify when primers straddle the breakpoint, as this region is presumably of a restrictive, wild type length of 22kb. B. Analysis of cDNA synthesized from total RNA extracted from control and proband’s dermal fibroblasts. PCR amplification of cDNA results in truncated transcript in proband. Lane 1 is 1Kb molecular weight DNA ladder. Arrows indicate position of forward and reverse primers used for cDNA amplification. Cloning of PCR product followed by sequencing showed direct splicing of exon 1 to downstream exon 8 in proband. C. Quantitative real time PCR analysis revealed significant reduced expression of SLC12A2 transcript normalized to POLR2A in proband fibroblasts compared to two independent fibroblast controls. D. Western blot analysis of protein lysates from fibroblasts probed with polyclonal anti-SLC12A2 (NKCC1) antibody shows no detectable SLC12A2 protein in proband compared to two independent controls. The lower panel shows western blotting with anti-GAPDH to confirm equivalent sample loading. E. Protein lysates from fibroblasts were immunoprecipitated with polyclonal anti-SLC12A2 (NKCC1) antibody conjugated to Protein A sepharose. Western blotting of immunoprecipitates with monoclonal anti-SLC12A2 (NKCC1) revealed the presence of SLC12A2 in both monomeric and dimeric forms in control while no SLC12A2 bands were detected in proband. Molecular weight in kilodaltons (kDa) is shown to the left of the panel.