exome sequencing identifies a novel homozygous mutation in the phosphate transporter slc34a1 in...

Exome Sequencing Identifies a Novel HomozygousMutation in the Phosphate Transporter SLC34A1 inHypophosphatemia and Nephrocalcinosis

Abbhirami Rajagopal, Débora Braslavsky, James T. Lu, Soledad Kleppe,Florencia Clément, Hamilton Cassinelli, David S. Liu, Jose Miguel Liern,Graciela Vallejo, Ignacio Bergadá, Richard A. Gibbs, Phillipe M. Campeau,and Brendan H. Lee

Department of Molecular and Human Genetics (A.R., D.L., R.G., P.C., B.L.), Baylor College of Medicine,One Baylor Plaza, Houston, Texas 77030; Centro de Investigaciones Endocrinológicas “Dr. CésarBergadá” (CEDIE) (D.B., F.C., H.C., I.B.), CONICET – FEI – División de Endocrinología, Hospital de NiñosRicardo Gutiérrez, C1425FD Buenos Aires, Argentina; Human Genome Sequencing Center (J.T.L., R.G.),Department of Structural and Computational Biology and Molecular Biophysics (J.T.L.), Baylor College ofMedicine, Houston, Texas 77030; Unidad de Metabolismo (S.K.), Hospital de Niños Ricardo Gutiérrez,C1425FD Buenos Aires, Argentina; Unidad de Nefrología (J.M.L., G.V.), Hospital de Niños Ricardo Gutiérrez,C1425FD Buenos Aires, Argentina; Howard Hughes Medical Institute (B.L.), Houston, Texas 77030

Context: Two Argentinean siblings (a boy and a girl) from a nonconsanguineous family presentedwith hypercalcemia, hypercalciuria, hypophosphatemia, low parathyroid hormone (PTH), andnephrocalcinosis.

Objective: The goal of this study was to identify genetic causes of the clinical findings in the twosiblings.

Design: Whole exome sequencing was performed to identify disease-causing mutations in the young-est sibling, and a candidate variant was screened in other family members by Sanger sequencing. Invitro experiments were conducted to determine the effects of the mutation that was identified.

Patients and Other Participants: Affected siblings (2 y.o. female and 10 y.o male) and their parentswere included in the study. Informed consent was obtained for genetic studies.

Results: A novel homozygous mutation in the gene encoding the renal sodium-dependent phos-phate transporter SLC34A1 was identified in both siblings (c.1484G�A, p.Arg495His). In vitro stud-ies showed that the p.Arg495His mutation resulted in decreased phosphate uptake when com-pared to wild-type SLC34A1.

Conclusions: The homozygous G�A transition that results in the substitution of histidine for ar-ginine at position 495 of the renal sodium-dependent phosphate transporter, SLC34A1, is involvedin disease pathogenesis in these patients. Our report of the second family with two mutatedSLC34A1 alleles expands the known phenotype of this rare condition. (J Clin Endocrinol Metab 99:E2451–E2456, 2014)

Renal phosphate absorption that occurs in the renalproximal tubules is a key process in the phosphate

homeostasis pathway. Two transporters sodium depen-

dent-phosphate transporter (NaPi-IIa) and NaPi-IIc, bothof which are localized to the apical membrane of mam-malian proximal tubules, mediate renal phosphate reab-

ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2014 by the Endocrine SocietyReceived February 20, 2014. Accepted July 14, 2014.First Published Online July 22, 2014

Abbreviations: NaPi-II, sodium dependent-phosphate transporter; PTH, parathyroidhormone.

J C E M O N L I N E

A d v a n c e s i n G e n e t i c s — E n d o c r i n e R e s e a r c h

doi: 10.1210/jc.2014-1517 J Clin Endocrinol Metab, November 2014, 99(11):E2451–E2456 jcem.endojournals.org E2451

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sorption (1–3). While in murine animal models, NaPi-IIa(SLC34A1) has been shown to be a key regulator of renalphosphate reabsorption; its role in phosphate metabolismin humans remains unclear (4). Although heterozygousSLC34A1 variants have been associated with renal phos-phate leak and hypercalciuria (5), it has been argued byothers that these variants might not play a causative rolein the disease pathogenesis (6, 7). A recent report by Ma-gen et al showed that a homozygous mutation (a 21 bpduplication) in SLC34A1 resulted in renal Fanconi’s syn-drome with proximal tubulopathy (8).

Here we report a single nucleotide change in SLC34A1identified by whole exome sequence analysis as a disease-causing variant in two siblings with hypercalcemia, hy-percalciuria, hypophosphatemia, and nephrocalcinosis.Phosphate uptake assays in HEK 293 (human embryonickidney) cells showed the inability of this novel SLC34A1variant to transport phosphate and thereby confirmingpathogenicity of SLC34A1 sequence variant.

Subjects and Methods

Subjects. We describe two Argentinean siblings (one boy and onegirl) from a nonconsanguineous family who presented with hy-percalcemia, hypercalciuria, hypophosphatemia, and nephro-calcinosis without proximal tubulopathy, renal failure nor skel-etal dysplasia (see pedigree in Figure 1).

Patient 1 is a male born at term with normal weight andheight. Nephrocalcinosis was found at 18 months in an ultra-sound performed due to repeated urinary tract infections. Hisclinical examination and skeletal radiographs were unremark-able. He had transient moderate hypercalcemia, which wasresolved at 2 years of age, transient hypophosphatemia thatrequired oral phosphate supplements, and has persistent hy-percalciuria. Parathyroid hormone (PTH) was initially undetect-able but became appropriate for serum calcium at 10 years ofage.

Patient 2 is a girl born at term, small for gestational age (birthheight �3.3 SDS) with breech presentation. Because of herbrother´s medical history, she was evaluated at 2 months of ageand was found to have nephrocalcinosis. She was under vitaminD prophylaxis (500 IU/d) with 25(OH)2D3 serum levels in theupper normal range and elevated 1,25(OH)2D3 levels. She hadsevere hypercalcemia with only transient response to medicaltreatment consisting of hyperhydration, diuretics, methylpred-nisone 1 mg/kg/d given twice and five IV pamidronate cycles.Hypercalcemia and low PTH resolved at 3.2 years of age. Hy-pophosphatemia progressively worsened and periodically requiredoral phosphate supplements for serum phosphate normalization.Nevertheless, hypercalciuria was persistently elevated through-out the follow-up period (there was only one normal urinarycalcium determination within glucocorticoid treatment). There-fore, potassium citrate therapy was instituted to prevent nephro-calcinosis progression. Patient 1 is growing appropriately, butpatient 2 has a severely short stature, without any sign of skeletaldisease or dysplasia on the radiographic skeletal survey. No bio-chemical or imaging abnormalities were identified in their par-ents; the only clinical sign of importance is the mother’s shortstature.

Whole exome sequencing. Exome sequencing was performedas described previously (9). Briefly, exomes were captured onNimblegen’s Baylor VCRome library (Roche NimbleGen) andsequencing was performed on the Illumina HiSeq 2000 platform(Illumina). Sequence reads were aligned to the hg18 referencehuman genome, SNPs were called, variants were annotated, andcandidate genes were assessed using various databases to predictexpression pattern, function, and potential pathogenic impact ofthe variants. The accession numbers for the human SLC34A1gene are as follows: NCBI Gene: 6569; GenBank transcript:NM_003052.4; CCDS protein: 4418.1.

Plasmids. hSLC34A1-pCMV Sport 6 plasmid was purchasedfrom the Harvard Plasmid repository. The arginine to histidinepoint mutation at position 495 was generated using theQuikChange II Site-Directed Mutagenesis Kit (Agilent Technol-ogies) as per the manufacturer’s protocol and confirmed by

sequencing.

Cell culture. HEK 293 cells were grownin DMEM containing 10% fetal bovineserum and 1% each of penicillin, strep-tomycin, and L-glutamine.

Real time PCR. Equal numbers of HEK293 cells were plated in 6-well plates.Cells were transfected with 2 �g DNAper well with 1:4 ratio of XtremeHP

transfection reagent (Roche). Forty eighthours after transfection, the growth me-dium was aspirated and the confluentmonolayer was rinsed with phosphatebuffered saline. Total RNA was extractedusing a Trizol reagent (Life Technologies).Samples were DNase (Roche) treated andthe Superscript III First Strand RT-PCR kit(Life Technologies) was used for cDNAsynthesis. The qRT-PCR was performed

Figure 1. Pedigree of the family. E�(c.[1484G�A];[1484G�A]) signifies that a DNA samplefrom the patient was evaluated and revealed the presence of the mutation in the homozygousstate. (c.[1484G�A];�) refers to a heterozygous mutation.

E2452 Rajagopal et al Homozygous SLC34A1 Mutation in NephrocalcinosisJ Clin Endocrinol Metab, November 2014, 99(11):E2451–E2456


on a LightCycler instrument (Roche). hGAPDH was used as theinternal control to normalize gene expression.

Immunofluorescence assay. Cells were transfected with 1 �gDNA per well with the XtremeHP transfection reagent (Roche) intwo-well chamber slides (Nunc). Briefly, cells were fixed withparaformaldehyde, permeabilized with triton X-100, blockedwith donkey serum plus bovine serum albumin. Primary an-tibody (rabbit anti-Npt2a antibody, Sigma) was incubated at1:75 dilution in 1% bovine serum albumin for 90 min. Afterwashing, samples were incubated with both alexa fluor 488wheat germ agglutinin and donkey-anti-rabbit conjugatedwith alexa fluor 594 for 1 hour. The slides were then washedand mounted with an antifade mounting reagent with4=,6-diamidino-2-phenylindole.

Phosphate uptake assay. Cells were transfected as describedabove and 48 h after transfection, then the confluent mono-layer was rinsed with an incubation buffer lacking the trans-port solute (10). Transport was then initiated by adding 1 mLof incubation buffer containing 1 �Ci/mL of 32P labeledmonobasic potassium phosphate (Perkin Elmer). The plateswere slowly agitated at 37°C. Cells were washed with ice-coldstop solution at 0, 5, 10, and 20 min to terminate the uptakeprocess. Cells were then solubilized with 0.5 mL triton X-100containing protease inhibitors. Radioactivity was measured in150 �L of the homogenate and total protein was measuredusing the Biorad protein assay.

Results

Clinical characteristics of the patientsThe results of clinical and biochemical evaluations of

the patients compared to the 21 bp duplication describedby Magen et al, are described in Table 1. The ultrasoundof the female child performed at 2 months of age showednephrocalcinosis and the radiographic images revealedrhizomelia at 4 months of age (Figure 2). At 3 years of age,the child did not show any signs of rickets or skeletal dys-plasia (Figure 2D).

A novel homozygous point mutation in SLC34A1 isidentified in the siblings

Whole exome analysis revealed a single G3A transi-tion that resulted in the substitution of histidine for argi-nine at position 495 of the sodium-phosphate trans-porter, SLC34A1 (Figure 3, A and D). The G3Asubstitution was further confirmed by Sanger sequenc-ing (Figure 3B). The high degree of conservation of thearginine residue across species (Figure 3C) and rarity ofthis variant in the population suggest that the mutationwe identified might play a causative role in defectivephosphate metabolism in this patient. A different variantat the same residue (rs199565633, p.R495C) was onlyobserved once, and in the heterozygous state, among 6503

individuals sequenced for this region in the Exome VariantServer release version v.0.0.22. (October 17, 2013).

Phosphate uptake is markedly reduced in cellsoverexpressing mutant SLC34A1

To test whether the p.Arg495His mutation resulted indysregulated phosphate transport, we performed phos-phate uptake assays using radioactive 32P in HEK 293 cellstransfected with wild-type or mutant SLC34A1 construct.Both wild type and mutant hSLC34A1 constructs are ex-pressed at similar levels and this expression was signifi-cantly higher than the control (Figure 3e). Immunofluo-rescence assays showed that both the wild-type andmutant proteins colocalize with wheat germ agglutinin tothe cell surface (Supplemental Figure 1). Forty-eight hourspost-transfection, cells were incubated with 1 �Ci/mL 32P-labeled potassium phosphate for 5, 10, or 20 min andphosphate uptake assay was performed as previously de-scribed (10). Cells overexpressing the wild type SLC34A1showed an increased phosphate uptake at all three timepoints as compared to cells expressing an empty vector(3F). Overexpression of mutant SLC34A1 did not result inan increased phosphate uptake and levels of radioactivephosphate remained similar to that of an empty vectorcontrol (Figure 3F).

Discussion

We have identified a homozygous single nucleotidevariation in SLC34A1 in siblings who presented withhypercalcemia, hypercalciuria, hypophosphatemia, andnephrocalcinosis. The causative role of the SLC34A1R495H mutation in the pathogenesis of the disease in ourpatients is supported by the following results: (a) the mu-tation segregates with the disease phenotype (b) the singlenucleotide variant we report is novel and a different vari-ant at the same residue is very rare (minor allele frequencyof 0.00008) (c) the arginine residue is highly conservedacross vertebrates and lies in the region that is thought tobe involved in PTH-mediated regulation of SLC34A1 and(d) in vitro phosphate uptake experiments showed a re-duction in the amount of phosphate transported by theNaPi-IIa R495H mutant.

TheroleofSLC34A1orNaPi-IIa inhumanrenalphosphatehomeostasis isnotwellunderstood(8,11).Recently,ahomozy-gous mutation was identified in SLC34A1 in two siblingsfrom a consanguineous family. While patients studied inthe Magen et al study have renal Fanconi syndrome andhypophosphatemic rickets, we did not detect these in ourpatients (8). Phenotypic heterogeneity is observed both inour patients, as evidenced by the few phenotypic signs in

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patient 1 as compared to patient 2, his sister, and also bythe difference with the other reported family though witha different mutation (8). We observe hypercalciuria butnot persistent hypercalcemia in our patients; this is con-sistent with results from previous studies showing that alow serum phosphate concentration could stimulate 1,25dihydroxy vitamin D synthesis, which, in turn could leadto hypercalciuria (12). Hypercalcemia has not been doc-umented previously, but it could be due to differences in

the age at which the patients were examined since in ourpatients, hypercalcemia was transient and spontaneouslyresolved at a young age (13). A study done by Chau et alshowed that the expression on NaPi-IIa is key for the de-velopment of nephrocalcinosis in the NaPi-IIa null mousemodel (14). They showed that Npt2a mRNA and proteinare expressed in the prenatal period and the onset ofnephrocalcinosis coincides with the absence of Npt2aexpression (14). Additionally, hypercalciuria second-

Table 1. Clinical and Biochemical Results

This Report

Homozygous Duplication, Magen D et al (8)Patient 1 (male) Patient 2 (female)

Mother FatherAdmission Last Visit Admission Last VisitPatient 1(male)

Patient 2(female)

ReferenceValues

Age (y) 1.5 10.3 0.24 3.2 N/A N/A 39 43 N/AHeight (Z-score) �1.2 �1.8 �3.1 �3.4 �2.0 �1.5 �4.1 �2.8 N/ADysmorphisms None None Narrow forehead,

thin lips,low-set ears

Narrow forehead,thin lips,low-set ears

No No No No N/A

Hypercalciuria Yes Yes Yes Yes No No In adolescencebut notadulthood

In adolescencebut notadulthood

N/A

Nephrocalcinosis Yes Yes Yes Yes No N/A No No N/ASkeletal

deformitiesNo No Rhizhomelia,

short neckRhizhomelia,

short neckNo No Bone pain, leg

deformities,osteomalacia

N/A N/A

Proximaltubulopathy

No No No No No N/A Yes Yes N/A

BMD (L1–L4)gm/cm2

N/A 0.864 N/A 0.651 N/A N/A N/A N/A N/A

Blood testsCalcium (mg/dL) 11 9.7 Between 11.5

and 14.79.8 9.8 9.3 9.9, previously

elevated9.1, previously

elevated8.5–10.8

Phosphate(mg/dL)

4.3 (Nl 4.3–5.4) 3.9 (Nl 3.7–5.4) 3.7 (Nl 4.3–5.4) 4.7 (Nl 4.3–5.4) 3.5 (Nl 2.5–4.5) 3.2 2 (Nl 2.5–4.5) 2.2 (Nl 2.5–4.5) Varies accordingto age andgender

Magnesium(mg/dL)

1.9 1.8 2 1.9 1.9 2 N/A N/A 1.7–2.3

Alkalinephosphatase(UI/L)

906 287 791 339 79 151 113 102 10–270

Creatinine(mg/dL)

0.38 0.7 0.3 0.3 0.78 0.89 1.5 0.9 0.5–1.3

Uric acid(mg/dL)

2.8 4.5 7.3 5.7 4.7 5.9 1.5 1.9 2.2–5.8

pH 7.42 7.36 7.38 7.39 7.36 N/A 7.36 7.37 7.38–7.41Bicarbonate

(mmol/L)22.4 22.5 23 24.2 26.2 N/A 24 25 22–26

Intact PTH(pg/mL)

Undetectable 19 Undetectable 12 22 24 45 29 8–54

25(OH)D3(ng/mL)

N/A 31 69 35.7 31,9 46.1 12.4 8.3 �30

1,25 (OH)2D3

(pg/mL)N/A N/A 213 N/A N/A N/A 21, previously

elevated12, previously

elevated20–71

FGF23sequencing

N/A N/A normal N/A N/A N/A N/A N/A N/A

GH tests (maxpeak-ng/mL)

N/A N/A N/A 10.1 N/A N/A N/A N/A �6

Urine testsGlucose

(mg/dL)N/A N/A N/A 90 (units) N/A N/A 100 300 N/A

TRP (%) N/A 88 90 91.6 N/A N/A 49 66 85–95Calcium:

creatinineratio

0.55 0.21 2.2 0.4 N/A N/A 0.19 0.1 �0.2

Protein(g/24 h)

N/A 0.09 N/A 0.04 N/A N/A 1.8 0.6 �0.2

Citrate N/A 2 mg/kg/24 h(Nl � 2)

2 mg/kg/24 h(Nl � 2)

2 mg/kg/24 h(Nl � 2)

N/A N/A 375 mg/24 h(Nl � 320)

420 mg/24 h(Nl � 320)

N/A

Abbreviation: N/A, not applicable.



ary to the increase in serum 1,25 dihydroxy vitamin D,is a risk factor for nephrocalcinosis in both rodent mod-els and humans (12).

The mutation we identified in SLC34A1, which re-sulted in the substitution of histidine for arginine at posi-

tion 495, resides in a domain thoughtto be essential for PTH-mediated en-docytosis of SLC34A1 (15). Parathy-roid hormone functions as a phos-phaturic hormone by inhibitingsodium-phosphate co-transport, in-ducing transporter internalizationand targeting the internalized trans-porter for lysosomal degradation(16–20).

In conclusion, we report a novelhomozygous single nucleotide muta-tion in SLC34A1 that plays a caus-ative role in the altered calcium phos-phate handling in this family. Thephenotypic difference from the pre-viously reported family highlightsthe clinical variability of this condi-tion. Exome sequencing, by provid-

ing detailed sequence information on most coding regions,is a powerful tool for research and the clinic. However,there are some limitations in the data that it can provide:some regions (such as GC rich regions) remain difficult to

Figure 2. Radiographic features of the female patient. A, Renal ultrasound at 2 months of age.B, x-ray at 3 months of age. C, rhizomelia at 4 months. D, x-ray at 3 years of age showing no signs ofrickets nor skeletal dysplasia. Growth arrest lines are consistent with bisphosphonate therapy.

Figure 3. Mutation in SLC34A1 results in decreased phosphate uptake in 93 cells. A, Results from whole exome analysis. B, Sanger sequencing of thepatients and parents. C, Conservation of the arginine residue among vertebrates. D, Model of heterozygous and homozygous mutations found inSLC34A1. E, Real time PCR performed in HEK 293 cells overexpressing either the empty vector, wild type or mutant hSLC34A1 constructs. Student’st-test was performed and the asterisk indicates significance at P � .01. F, Phosphate uptake assay performed in 293 cells overexpressing either the emptyvector (diamonds), wild type (squares), or mutant SLC34A1 (triangles) constructs. Student’s t-test was performed and the asterisk indicates significance atP � .05, compared to the vector alone. The uptake assay was performed three separate times and each assay was done in duplicate.

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sequence, genes with other homologous sequences (eg,paralogs, pseudogenes) are difficult to map, the sensitivityfor whole exon deletions is variable, and by definition itdoes not cover noncoding regions which could affect geneexpression (promoters and enhancers, deep-intronic splic-ing regulators). Nevertheless, in the field of endocrinol-ogy and metabolism, it has the potential to identify newdisease genes, to provide a rapid genetic diagnosis forknown diseases, and to expand the phenotypic variabil-ity of previously described disorders.

Acknowledgments

We thank Shalini N Jhangiani for exome sequencing coordination,Yuqing Chen and Terry Bertin for technical support, and ErichFradinger (IDIM) for 1,25 dihydroxy vitamin D determinations.

Address all correspondence and requests for reprints to:Brendan Lee, Department of Molecular and Human Genetics,Baylor College of Medicine, One Baylor Plaza, MS BCM225,Houston, Texas 77030. E-mail: [email protected].

This work was supported by the Baylor College of Medicine(BCM) Intellectual and Developmental Disabilities ResearchCenter (HD024064) from the Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development, the BCM Ad-vancedTechnologyCoreswithfundingfromtheNational Institutesof Health (NIH) (AI036211, CA125123, and RR024574), the Ro-lanette and Berdon Lawrence Bone Disease Program of Texas, theBCM Center for Skeletal Medicine and Biology, National Insituteof Diabetes and Digestive and Kidney Diseases training Grant No.5T32DK060445-10 (A.R.) and NIH Grant No. HD070394 (B.L.).

The ethics committee approved our study protocol and writ-ten informed consent was obtained from the parents.

Disclosure Summary: The authors have nothing to disclose.

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