JOURNAL READING

Patients with autoinflammatory disease sometimes present with clinical findins that encompass
multiple oran systems!" #hree unrelated children presented to the National Institutes of $ealth
%NI$& 'linical 'enter with intermittent fe(ers) recurrent lacunar stro*es) ele(ated le(els of acute+
phase reactants) li(edoid rash) hepatosplenomealy) and hypoammalo,ulinemia! 'ollecti(ely)
these findins do not easily fit with any of the *nown inherited autoinflammatory diseases!
$ereditary or ac-uired (ascular disorders can ha(e protean manifestations yet ,e caused ,y
mutations in a sinle ene! Diseases such as the Aicardi.Gouti/res syndrome)0)1 polypoidal
choroidal (asculopathy)2 sic*le cell anemia)3 li(edoid (asculopathy)4 and the small+(essel
(asculitides5)6 are e7amples of systemic diseases that may present with su,stantial (ascular
patholoic findins! In some cases) these disorders are not dianosed until a central ner(ous
system e(ent occurs! 8uch potentially catastrophic neuroloic e(ents often prompt the search for
other coincidental findins that may ena,le a definiti(e dianosis! 9e used whole+e7ome
se-uencin to screen for a common enetic cause of disease in these patients! Our in(estiations
led to the disco(ery of a pathway lin*in inflammation with (ascular de(elopment!
METHODS
Patients
9e e(aluated the initial three patients and their unaffected parents) as well as three patients with
a similar phenotype) two youn si,lins with polyarteritis nodosa) and one patient with small+
(essel (asculitis! :i(e of the patients were e(aluated at the NI$ 'linical 'enter) and the other
four at the Great North 'hildren;s $ospital in the United <indom and the $acettepe Uni(ersity
'hildren;s $ospital in #ur*ey! All the patients were enrolled in a study that had ,een appro(ed ,y
the respecti(e institutional re(iew ,oard! All the patients or their parents pro(ided written
informed consent! All the authors (ouch for the accuracy and completeness of the data and
analyses reported and for the fidelity of the study to the protocol! Lacunar stro*es were defined
accordin to standard criteria!=
Genetic and Functional Analysis
9e performed whole+e7ome se-uencin in the initial three patients and their unaffected parents
and candidate+ene se-uencin in the other si7 patients! En>yme assays) immuno,lottin)
immunohistochemical testin) flow cytometry) and cyto*ine profilin were performed on
samples from the patients! #o study protein function) we used morpholino+mediated *noc*downs
in >e,rafish and short hairpin RNA %shRNA& *noc*downs in U=15 cells cultured with human
dermal endothelial cells! #he 8upplementary Appendi7 %a(aila,le with the full te7t of this article
at NEJ?!or& descri,es the methods used for all these procedures!
RESULTS
Clinical Phenotype
Initially) three patients with recurrent fe(ers and early+onset stro*e were e(aluated@ the first
patient presented to the NI$ at 1 years of ae) and the other two at 4 years of ae! After they had
underone whole+e7ome se-uencin) two additional patients with a similar phenotype were
recruited! All fi(e patients were unrelated) with no history of consanuinity) and represented
sporadic cases of European ancestry %i!e!) were not in a founder population&@ all the patients
underwent e7tensi(e clinical assessment %#a,les 8" throuh 82 in the 8upplementary Appendi7&!
#he parents and si,lins of the patients from all fi(e families were healthy! All the patients had
presented with recurrent fe(ers and li(edo racemosa %:iure "AFIGURE
'linical :indins and Pedirees of Patients with Deficiency of Adenosine Deaminase 0
%ADA0&!& in early childhood! 8*in ,iopsies re(ealed a predominance of neutrophils and
macrophaes in the interstitium) with peri(ascular # lymphocytes) without fran* (asculitis %:i!
8"A in the 8upplementary Appendi7&! In one patient) necroti>in (asculitis was present in the
deep dermis %:iure "A and "') and :i! 8"A) 8"') and 8"D in the 8upplementary Appendi7&!
Durin fe,rile episodes) the le(els of acute+phase reactants were mar*edly ele(ated in all the
patients!
Each patient had had stro*es ,efore 3 years of ae %#a,les 8" and 81 in the 8upplementary
Appendi7&! #he stro*es occurred mainly durin episodes of inflammation) althouh fe(er was
not always o,ser(ed! ?anetic resonance imain %?RI& of the ,rain showed e(idence of acute
or chronic small su,cortical infarcts"B in(ol(in the deep+,rain nuclei and the ,rain stem) which
are consistent with small+(essel occlusions %lacunar stro*es&=)"B %:iure "D) "E) and ":) and
:i! 80A) 80A) and 80' in the 8upplementary Appendi7&! As o,ser(ed on ?RI) the su,cortical
white matter was spared! #wo patients who underwent e7amination of the cere,rospinal fluid
durin acute e(ents had mild lymphocytic pleocytosis!
8e(eral stro*e e(ents were hemorrhaic or underwent hemorrhaic transformation) leadin to
disa,lin se-uelae in some patients %:iure "G) and :i! 80D) 80E) and 80: in
the 8upplementary Appendi7&! #he interpretation of intracranial ,leedin as a phenotypic feature
was complicated ,y the concomitant use of antiplatelet aents) warfarin) or ,oth) althouh small)
deep hemorrhaes are increasinly reconi>ed as ,ein within the spectrum of lacunar
disease!"B)"" In all fi(e patients) manetic resonance anioraphy showed no e(idence of
cere,ral (asculitis@ the a,sence of cere,ral (asculitis was corro,orated ,y con(entional
anioraphy in three patients! 'omputed tomoraphy re(ealed no calcifications of the ,asal
anlia! In two patients who underwent ,iopsy of the ,rain) there was prominent e7tra(asation of
erythrocytes into the Circhow.Ro,in spaces and white matter around small (essels) without
clinically sinificant inflammation %:iure "$&! #hree patients had ophthalmoloic in(ol(ement
%#a,le 8" in the 8upplementary Appendi7&!
:our patients presented with hepatosplenomealy %:i! 81A) 81A) and 81' in the 8upplementary
Appendi7&! One patient had portal hypertension) with hepatofual flow ,etween the left portal
and um,ilical (eins %:i! 81D in the 8upplementary Appendi7&! Li(er ,iopsy re(ealed
endotheliali>ation of the hepatic sinusoids %:i! 81E and 81: in the 8upplementary Appendi7&!
:our patients presented with hypoammalo,ulinemia) and two had recurrent ,acterial and (iral
infections ,efore immunosuppressi(e treatment was initiated! :our patients had (aryin derees
of lymphopenia! I? le(els were consistently low in all fi(e patients %#a,le 82 in
the 8upplementary Appendi7&!
$ypertension) cardioenic em,olism) and dia,etes were ruled out) and e7tensi(e hematoloic
studies did not show hypercoaula,ility! At the onset of stro*es) all the patients were neati(e for
antiphospholipid anti,odies %#a,le 82 in the 8upplementary Appendi7&! O(er time) lupus
anticoaulant de(eloped in four patients! $ih doses of lucocorticoids only partially controlled
fe(er) rash) and acute+phase reactants! Despite aressi(e treatment with lucocorticoids)
cyclophosphamide) and cyto*ine inhi,itors) Patient 2 had her most serious e(ent at 01 years of
ae!
CECR1 Mutations
9hole+e7ome se-uencin was performed in Patients " and 0 and their unaffected parents %:iure
"I&! After filterin for no(el and rare (ariants %allele fre-uency) D"E&) we identified
appro7imately "5BB candidate (ariants in each trio! 9e hypothesi>ed that the disorder could ,e
caused ,y either de no(o or recessi(e mutations! A sinle common candidate ene was identified
only under the recessi(e model %:i! 82A in the 8upplementary Appendi7&! Aoth patients were
compound hetero>yous for missense mutations in CECR1, encodin ADA0 %:i! 82A in
the 8upplementary Appendi7&) and shared the p!#yr231'ys mutation! All four parents were
carriers@ the unaffected si,lin in :amily " was a noncarrier) and ,oth unaffected si,lins in
:amily 0 were carriers! 9e confirmed all (ariants ,y means of 8aner se-uencin %:i! 82' in
the 8upplementary Appendi7&!
9e also performed whole+e7ome se-uencin in Patient 1! #he only ene in common with a ene
in Patient " or Patient 0 that e7hi,ited a deleterious mutation was CECR1! Patient 1 was
hetero>yous for the p!Ar"4=Gln (ariant) which had ,een inherited from his father! A hih+
density sinle+nucleotide polymorphism array identified a 06+*, deletion in Patient 1 and his
mother that included the 3F untranslated reion and e7on " of CECR1! #he deletion ,rea*point
was confirmed ,y means of enomic se-uencin %:i! 82' in the 8upplementary Appendi7&!
'omplementary DNA se-uencin showed that the maternal allele carryin the deletion was not
e7pressed! 'andidate+ene se-uencin in Patients 2 and 3 identified one no(el (ariant)
p!$is""0Gln) and three mutations pre(iously found in Patients ") 0) and 1 %:iure "I&! #hree of
the fi(e missense CECR1 mutations were shared amon these fi(e patients!
9e su,se-uently performed candidate+ene se-uencin on samples from four additional patients
from other centers %:iure "I) and #a,le 83 in the 8upplementary Appendi7&! Patient 4) a "3+
year+old ,oy from the United <indom) had recurrent fe(er) lacunar stro*es) and cutaneous
manifestations that were different from those o,ser(ed amon the patients at the NI$ %:i! 83A
and 83A in the 8upplementary Appendi7&@ he died in 0B"1 from complications of his disease! $e
was compound hetero>yous for the p!?et"#hr null allele and p!Ile=1#hr! Patients 5 and 6 were
si,lins from #ur*ey with a history of stro*es who met the criteria for polyarteritis nodosa and
were referred on the ,asis of the wor* ,y Na(on El*an and colleaues) now reported in
the Journal!"0#hese patients) as well as a #ur*ish patient with small+(essel (asculitis ,ut no
history of stro*e %:i! 83' and 83D in the 8upplementary Appendi7&) were homo>yous for the
p!Gly25Ar mutation %:iure "I) and :i! 82' in the 8upplementary Appendi7&! Althouh the
p!Gly25Ar (ariant has not ,een reported in Europeans) we found that the carrier fre-uency of
this (ariant in #ur*ish controls was B!BB0! #he *ey clinical features of all nine patients seen at
the NI$ and outside centers are summari>ed in #a,le "TA!LE 'linical and
La,oratory ?anifestations in Patients with Deficiency of Adenosine Deaminase 0!!
'onser(ed haplotypes suested common ancestries for the shared p!Gly25Ala) p!Gly25Ar)
p!Ar"4=Gln) and p!#yr231'ys mutations %:i! 84 in the 8upplementary Appendi7&! #hree of
these mutations were found at low fre-uencies in the "BBB Genomes ProGect) the GO E7ome
8e-uencin ProGect %E8P43BB& of the National $eart) Lun) and Alood Institute) and the 'lin8e-
ProGect %#a,le 84 in the 8upplementary Appendi7&! All the (ariants were computationally
predicted to ,e li*ely to disrupt function %#a,le 84 in the 8upplementary Appendi7&) and they
affect e(olutionarily conser(ed residues %:i! 85A in the 8upplementary Appendi7&! 9e
e7amined indi(idual+le(el data from the E8P43BB to e7plore the CECR1 mutations reported
there! In the 8i,lins with Ischemic 8tro*e 8tudy %89I88&"1su,cohort that included =2 patients
with late+onset stro*e %Genotypes and Phenotypes data,ase) National 'enter for Aiotechnoloy
Information accession num,er) phsBBB105!("!p"!c"&) 0 ,rothers with ischemic lacunar stro*es
were hetero>yous carriers for the p!#yr231'ys mutation in ADA0 that was seen in 1 of our
pediatric patients %:i! 86 in the8upplementary Appendi7&!
Functional Studies o" Mutant ADA#
ADA0 has partial structural homoloy with human ADA"! Aoth proteins con(ert adenosine to
inosine and 0F+deo7yadenosine to 0F+deo7yinosine) ,ut the affinity of ADA0 for adenosine is
lower than that of ADA" ,y a factor of appro7imately "BB! 9hereas ADA" is monomeric and
larely intracellular) ADA0 is dimeric and secreted! Inherited ADA" deficiency causes profound
lymphopenia and se(ere com,ined immunodeficiency disease %8'ID&) which is associated with
the to7ic intracellular accumulation of deo7yadenosine nucleotides!"2
'omputer modelin of the missense mutations presented here) which was ,ased on the crystal
structure of human ADA0)"3 suests that these mutations are pro,a,ly loss+of+function
mutations %:i! 85A and 85' in the 8upplementary Appendi7&) affectin the catalytic and
dimeri>ation domains and protein sta,ility! Usin two independent assays) we measured ADA0
acti(ity in plasma and serum samples from the patients at the NI$) their relati(es) and healthy
adult and pediatric controls! #he patients had sinificantly diminished ADA0 acti(ity in plasma
%:iure 0AFIGURE # E(idence of Loss+of+:unction ?utations in ADA0!&!
9estern ,lot analysis showed that the ADA0 protein was reduced in cell lysates and a,sent from
the supernatants of macrophaes from patients from whom cultures were o,tained %:i! 8= in
the 8upplementary Appendi7&!
In assays measurin ADA acti(ity in monocytes and plasma from the patients) a sinificant
reduction was o,ser(ed only in ADA0+specific acti(ity) whereas ADA"+specific acti(ity was
preser(ed %:i! 8"BA and 8"BA and #a,le 85 in the 8upplementary Appendi7&! #he results of
thin+layer chromatoraphic assays for ADA" acti(ity in erythrocyte hemolysates were normal)
and the accumulation of deo7yadenosine nucleotides) which is the ,iochemical hallmar* of
ADA" deficiency) was a,sent in erythrocytes from patients with ADA0 deficiency %:i! 8"B'
and 8"BD in the 8upplementary Appendi7&!
I$$unolo%ic Assess$ent o" ADA# De"iciency
E7tensi(e studies of serum cyto*ines or of cyto*ines produced ,y cultured peripheral+,lood
mononuclear cells did not show any con(incin differences ,etween findins in the patients and
those in healthy controls %:i! 8"" in the 8upplementary Appendi7&! Gi(en the profound #+cell
defects in ADA" deficiency)"4 we e7amined thymic output and #+cell function! Recent thymic
emirants %'D1H) 'D1"H) and 'D23RAH& and nai(e # cells %'D1H) 'D40LH) and 'D23RAH&
were normal %data not shown&! Patients with ADA0 deficiency had normal short+term #+cell
acti(ation and normal proliferati(e responses to anti+'D1 anti,odies %:i! 8"0 in
the 8upplementary Appendi7&! #hey had slihtly increased proliferation in ,oth 'D2H and 'D6H
su,sets in response to phytohemalutinin stimulation ,ut normal production of effector
cyto*ines!
9hen peripheral+,lood mononuclear cells were cultured for 26 hours without stimulation) hiher
rates of spontaneous A+cell death were seen in the samples from the patients than in the samples
from healthy controls %:i! 8"1 in the 8upplementary Appendi7&! As compared with samples
from ae+matched controls) samples from patients had fewer memory A cells in the peripheral
,lood) lower e7pression of 'D05 and IG on A cells after induction with a (ariety of stimuli) and
a modest reduction in the terminal differentiation of A cells and immunolo,ulin+secretin cells
after #+cell+dependent stimulation %:i! 8"2 in the 8upplementary Appendi7&! A ,one marrow
aspirate and ,iopsy performed in one patient re(ealed normal A+cell maturation ,ut reduced
num,ers of 'D"16H plasma cells %data not shown&!
An Ani$al Model o" ADA# De"iciency
Althouh there is no murine ortholoue of CECR1, there are two paralos in
>e,rafishI cecr1a%chromosome 03& and cecr1b %chromosome 2&! A cecr1a hypomorphic line
enerated ,y means of random insertional mutaenesis with a retro(iral (ector %:i! 8"3A)
8"3A) and 8"3' in the8upplementary Appendi7& had no o(ert phenotype! 9e de(eloped a
transient *noc*down stratey for cecr1b usin translation+,loc*in and splice+,loc*in
morpholinos %:i! 8"4A in the8upplementary Appendi7&! A re(erse+transcriptase.polymerase+
chain+reaction assay of em,ryos inGected with morpholino antisense strateies to pre(ent the
proper splicin of e7on 1 %E1I1+?O& confirmed the a,ility of the antisense construct to induce
s*ippin of e7on 1 in the cecr1btranscript %:i! 8"4A in the 8upplementary Appendi7&) resultin
in a frameshift) premature termination) and a reduced transcript le(el of 6E of nonmutant
e7pression %:i! 8"3' in the8upplementary Appendi7&!
9e o,ser(ed intracranial ,leedin %:iure 0A& with ,oth cecr1b morpholinos in a dose+
dependent manner %:i! 8"4' in the 8upplementary Appendi7&) which suested the presence of
defects in (essel de(elopment or interity!"5 Alood+(essel morpholoic features appeared to ,e
normal) despite e(idence of hemorrhae and ischemia in em,ryos inGected with cecr1b+specific
morpholino olionucleotides taretin the translation initiation site %A#G+?O& %:i! 8"4D in
the 8upplementary Appendi7&! Intracranial hemorrhae was ,loc*ed ,y coinGection with
nonmutant human CECR1messener RNA %mRNA& ,ut not with mutant transcripts %:i! 8"4E
in the 8upplementary Appendi7&!
Je,rafish that were se(ere hypomorphs for cecr1a showed no sinificant increase in intracranial
,leedin@ in addition) when em,ryos that were hypomorphs for cecr1a were inGected
with cecr1bmorpholinos) there was no sinificant increase in intracranial ,leedin in these
em,ryos as compared with their morpholino+inGected nonmutant si,lins %data not shown&)
suestin thatcecr1b primarily pro(ides the ADA0 function necessary to pre(ent the
hemorrhain phenotypes! #he two paralos showed different e7pression patterns) suestin
that they ha(e functionally di(ered %:i! 8"3D and 8"3E in the 8upplementary Appendi7&!
9hen A#G+?O was used to disrupt cecr1b e7pression in mpxIEG:P transenic >e,rafish %with
the ene for enhanced reen fluorescent protein under the control of a myeloid+specific
pero7idase promoter&) in which neutrophils are la,eled reen) there was mar*ed neutropenia
%:i! 8"4: in the8upplementary Appendi7&! #his phenotype was ,loc*ed ,y the coinGection of
nonmutant humanCECR1 mRNA ,ut not ,y the coinGection of $""0K or R"4=K mRNA) ,oth of
which are associated with stro*e in our patients!
E""ects o" ADA# De"iciency on Endothelial and Leu&ocyte De'elop$ent
Gi(en the >e,rafish data) we e7amined endothelial interity and acti(ation in patients! 8tainin
with anti+'D1" anti,odies showed su,stantial endothelial damae in ,iopsy specimens from the
,rain %:i! 8"5A) 8"5D) 8"5J) and 8"5? in the 8upplementary Appendi7& and from lesional
s*in %:i! 8"5A) 8"5') 8"5E) 8"5:) 8"5<) 8"5L) 8"5N) and 8"5O in the 8upplementary
Appendi7&! Endothelial+cell acti(ation as shown ,y E+selectin stainin was o,ser(ed ,oth in
,rain+,iopsy specimens %:iure 1AFIGURE ( Effect of ADA0 Deficiency in
Patients!) and :i! 8"5G in the 8upplementary Appendi7& and in s*in+,iopsy specimens %:iure
1A and 1') and :i! 8"5$ and 8"5I in the8upplementary Appendi7&! #here was also increased
stainin for interleu*in+"L %:iure 1D) 1E) and 1:&) induci,le nitric o7ide synthase %:i! 8"6A)
8"6A) and 8"6' in the 8upplementary Appendi7&) and tumor necrosis factor M %:i! 8"6D) 8"6E)
and 8"6: in the 8upplementary Appendi7&) indicatin inflammation!
CECR1 is not e7pressed) nor is the ADA0 protein detecta,le) in cultured human endothelial cells
%:i! 8"= in the 8upplementary Appendi7&! #his implicates additional cell types in the
(asculopathy! Pre(ious studies ha(e shown a role for human ADA0 in the differentiation of
monocytes to macrophaes!"6 Usin shRNA constructs to silence the e7pression of ADA0 in
myeloid U=15 cells) we o,ser(ed mar*ed impairment of macrophae differentiation induced ,y
phor,ol myristate acetate %:i! 80BA) 80BA) and 80B' in the8upplementary Appendi7&! Under
standard conditions)"= monocytes from the patients differentiated into ?" macrophaes ,ut
poorly differentiated into ?0 macrophaes %:iure 1G throuh J) and :i! 80" in
the 8upplementary Appendi7&) which may ha(e proinflammatory effects!0B)0" :urthermore)
shRNA *noc*down of ADA0 in U=15 cells led to considera,le disruption of cocultured
monolayers of human primary dermal micro(ascular endothelial cells %:i! 800A in
the 8upplementary Appendi7&) as did monocytes from two patients with ADA0 deficiency
%:iure 1< and 1L) and :i! 800A in the 8upplementary Appendi7&!
DISCUSSIO)
9e descri,e a disorder characteri>ed ,y recurrent fe(ers) a spectrum of (ascular patholoic
features) and mild immunodeficiency! #he data implicate loss+of+function mutations
in CECR1,encodin the ADA0 protein %#a,le 86 in the 8upplementary Appendi7&! 8tudies in
patients; cells and in >e,rafish support the hypothesis that ADA0 is a rowth factor for
endothelial and leu*ocyte de(elopment and differentiation! As compared with patients with
ADA" deficiency) those with ADA0 deficiency ha(e only a mild immunodeficiency that is most
e(ident in A cells!
Our data corro,orate a role for ADA0 as the prototype for a family of adenosine deaminase.
related rowth factors) a role that had pre(iously ,een suested ,y studies in 7enopus and
drosophila!00+02 #he e7periments descri,ed here indicate that cecr1b is essential for ,oth
(ascular interity and neutrophil de(elopment in the >e,rafish em,ryo and that ,oth phenotypes
are pre(ented ,y nonmutant) ,ut not ,y mutant) human CECR1 mRNA!
?oreo(er) althouh ADA0 is not e7pressed in endothelial cells) there is a defect in endothelial
interity in the small (essels of patients with ADA0 mutations as well as impairment of ?0
macrophae differentiation! ADA0 is *nown to ,e produced ,y myeloid cells and to promote
macrophae differentiation!"6 #oether) the data from >e,rafish and patients suest that ADA0
deficiency may compromise endothelial interity while polari>in macrophae and monocyte
su,sets toward proinflammatory cells) esta,lishin a (icious circle of (asculopathy and
inflammation!0B)0")03
Gi(en the spectrum of disease o,ser(ed in these nine patients) ADA0 may play a role in other
disorders! #he o,ser(ation of two ,rothers who were hetero>yous for the p!#yr231'ys mutation
and who had late+onset lacunar stro*es warrants larer+scale se-uencin studies to e7plore the
sinificance of CECR1 (ariants in comple7 forms of lacunar stro*e and (asculitis! It is also
possi,le that ADA0 deficiency accounts for some patients with 8neddon;s syndrome) a poorly
understood disorder that is most common in middle+ae women and that is characteri>ed ,y
li(edoid rash and stro*e) with antiphospholipid anti,odies present in some of the patients!04
#herapeutic strateies for the treatment of patients with ADA0 deficiency re-uire in(estiation!
8ince the phenotype appears not to ,e caused ,y the accumulation of adenosine and
deo7yadenosine) treatment with peylated ADA") an effecti(e therapy for ADA+related 8'ID) is
unli*ely to ,e successful! $owe(er) since ADA0 is found in plasma) these patients may ,enefit
from fresh+fro>en plasma or recom,inant ADA0) assumin that cell.cell interactions are not
necessary for effecti(e ADA0 acti(ity! Alternati(ely) i(en that monocytes and macrophaes) the
main producers of ADA0) are deri(ed from ,one marrow) it is possi,le that ,one marrow
transplantation or enetic manipulation of ,one marrow cells has a role in the treatment of these
patients!
In conclusion) we defined a enetic disorder) for which we propose the name Ndeficiency of
ADA0O %DADA0&) that connects systemic inflammation) (ascular patholoy) and mild
immunodeficiency! DADA0 esta,lishes a role for adenosine deaminase.related rowth factors in
human disease and pro(ides potential dianostic and therapeutic strateies for a newly
reconi>ed roup of patients!
8upported ,y the National Institutes of $ealth %NI$& Intramural Research Prorams) includin
the Intramural Research Prorams of the National $uman Genome Research Institute) the
National Institute of Arthritis and ?usculos*eletal and 8*in Diseases) the National $eart) Lun)
and Alood Institute %N$LAI&) the National Institute of Allery and Infectious Diseases) the
National Institute of Dia,etes and Diesti(e and <idney Diseases) the National 'ancer Institute)
the Undianosed Diseases Proram of the 'ommon :und of the Office of the Director of the
NI$) and the NI$ 'linical 'enter@ and ,y rants from 8ima #au Pharmaceuticals %to Dr!
$ershfield& and the :innish Academy %to Drs! Andrey and Anton Ja(ialo(&!
Disclosure forms pro(ided ,y the authors are a(aila,le with the full te7t of this article at
NEJ?!or!
Drs! Jhou and Pan and Drs! Aoehm) <astner) and A*sentiGe(ich contri,uted e-ually to this
article!
#his article was pu,lished on :e,ruary "=) 0B"2) at NEJ?!or!
9e than* the staff at the NI$ Intramural 8e-uencin 'enter for performin whole+e7ome
se-uencin) Dr! Leslie Aiesec*er for sharin (ariant data from the 'lin8e- ProGect) ?r! 8tephen
9inco(itch for performin confocal microscopy of >e,rafish) ?s! 8unny $uan for em,ryo
inGections) Dr! Eyal ?uscal for clinical support and e7pertise) Drs! Ro,ert I! Richards and Arant
?! 9einstein for a critical readin of an earlier (ersion of the manuscript and for helpful
discussion) Dr! 'hristian A! 'om,s and Dr! Daniela ?alide %Liht ?icroscopy 'ore :acility)
N$LAI) NI$& for professional s*ills and ad(ice reardin microscopy+related e7periments) Dr!
Ju+Qi Pu and colleaues at the N$LAI Patholoy 'ore :acility for professional s*ills and
ad(ice) and all the affected children and their families) and the healthy children and adult
controls) for participation in this research study!