REPORT Identification of Mutations in SLC24A4, Encoding a Potassium-Dependent Sodium/Calcium Exchanger, as a Cause of Amelogenesis Imperfecta David A. Parry, 1,6 James A. Poulter, 1,6 Clare V. Logan, 1 Steven J. Brookes, 2 Hussain Jafri, 1,3 Christopher H. Ferguson, 4 Babra M. Anwari, 5 Yasmin Rashid, 3 Haiqing Zhao, 4 Colin A. Johnson, 1 Chris F. Inglehearn, 1 and Alan J. Mighell 1,2, * A combination of autozygosity mapping and exome sequencing identified a null mutation in SLC24A4 in a family with hypomineralized amelogenesis imperfect a (AI), a condition in which tooth enamel formation fails. SLC24A4 encodes a calcium transporter upregulated in ameloblasts during the maturation stage of amelogenesis. Screening of further AI families identified a missense mutation in the ion- binding site of SLC24A4 expected to severely diminish or abolish the ion transport function of the protein. Furthermore, examination of previously generated Slc24a4 null mice identified a severe defect in tooth enamel that reflects impaired amelogenesis. These findings support a key role for SLC24A4 in calcium transport during enamel formation. The formation of dental enamel (amelogenesis) results in the hardest, most mineralized tissue in the body. Amelo- genesis is completed before tooth eruption and thereafter it has no capacity for cellular repair. Mature enamel consists almost exclusively of highly organized, calcium hydroxyapatite (Ca 10 [PO 4 ] 6 [OH] 2 ) crystals, which form in a discrete extracellular compartment within the devel- oping tooth. 1 Ameloblasts, the enamel forming cells within the enamel organ, secrete an organic matrix and regulate the mineralization of enamel via several mecha- nisms that include the secretion of matrix proteins, which act as potential modulators of crystal growth, temporospa- tial control of protease secretion to sequentially degrade matrix proteins, removal of degraded protein from the enamel matrix, and control of mineral ion transport to accommodate secondary crystal growth during the matu- ration stage of enamel development. 2 Failure of amelogenesis presents clinically as amelogene- sis imperfecta (AI [MIM %104530]), a genetically and phenotypically heterogeneous group of inherited condi- tions with a prevalence ranging from 1/700 to 1/ 14,000. 3,4 AI may be classified as either hypoplastic AI, in which the enamel volume is diminished, or hypomineral- ized AI, characterized by variable degrees of incomplete mineralization of the enamel matrix, typically with a near-normal enamel matrix volume prior to posteruptive changes and premature failure. The negative psychological impact of AI on affected individuals and their families can be profound and the dental treatment required is chal- lenging. 5 Mutations in genes encoding enamel matrix proteins 6,7 and mutations in genes encoding proteases that digest enamel matrix proteins 8,9 have been identified as causes of AI. However, little is known about the function of genes identified in recent genetic studies. 10–17 Surprisingly, considering the essential role for calcium transport across the enamel organ in amelogenesis, 18 to date no known calcium transport proteins have been implicated in the pathogenesis of AI. We identified a consanguineous family (AI-112) from Pakistan segregating autosomal-recessive hypomineralized AI in the absence of other health problems (Figure 1; Figure 2A). The study was performed according to the prin- ciples of the declaration of Helsinki with ethical approval and family participation following informed consent. Individuals IV:1, IV:5, and IV:6 were analyzed using Affymetrix 6.0 SNP microarrays and common regions of homozygosity identified using AutoSNPa. 19 Five regions of homozygosity spanning approximately 16 Mb were identified (see Table S1 available online), none of which overlapped with previously published AI loci. We therefore performed exome sequencing on DNA from individual IV:5 using a SureSelect All Exon V4 reagent (Agilent Tech- nologies, Edinburgh UK). Three micrograms of genomic DNA were processed according to the Agilent SureSelect Library Prep protocol. Sequencing was performed using a 150 bp paired-end protocol on an Illumina MiSeq sequencer. The resulting sequence reads were aligned to the human reference sequence (GRCh37) using Novoalign software (Novocraft Technologies, Selangor, Malaysia). This alignment was processed in the SAM/BAM format 20 using Picard and The Genome Analysis Toolkit (GATK) 21,22 java programs in order to correct alignments around indel sites and mark potential PCR duplicates. Following postprocessing and duplicate removal a mean depth of 25.55 reads was achieved for targeted exons in 1 Leeds Institute of Molecular Medicine, St James’s University Hospital, University of Leeds, LS9 7TF Leeds, UK; 2 Leeds Dental Institute, University of Leeds, LS2 9LU Leeds, UK; 3 Gene Tech Lab 146/1, Shadman Jail Road, Lahore 54000, Pakistan; 4 Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA; 5 de’Montmorency College of Dentistry, Lahore 54000, Pakistan 6 These authors contributed equally to this work *Correspondence: a.j.mighell@leeds.ac.uk http://dx.doi.org/10.1016/j.ajhg.2013.01.003. Ó2013 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 92, 307–312, February 7, 2013 307