Peripheral Blood Mononuclear Cells Express Mutated NCCT mRNA in ...

Peripheral Blood Mononuclear Cells Express Mutated NCCT mRNA in Gitelman’s Syndrome: Evidence for Abnormal Thiazide-Sensitive NaCl Cotransport NATALIA

ABULADZE,*

NORIMOTO

YANAGAWA,t

IVAN

DEBRA NEWMAN,* JAMES HWANG,* KO!CHI ALEXANDER PUSHKIN,* ROBERT L. MODLIN, *Di,,isio,i

of Nephrologv,

Administration,

Abstract.

Genetic

between porter

gene

Several study

(NCCT

or TSC) NCCT

was

performed

mononuclear

cells

their

in GS.

revealed

and

that

quenee

of

PBMC

was

ferent

PBMC

NCCT

mutations

(compound maternal

were

exons

3 and

syndrome

pokalemia;

hypomagnesemia;

wasting;

OS

and

(SLCI2A3)

findings

groups

(4-8).

studies,

combined

encodes

cotransporter against

with

PBMC. from

patients’

GS,

NCCT

(GS)

tubule

et

a!.

disorder subsequently

In situ

hybridization

with

the

apical

(9-12). I intensely

the

presence

inhibited

22Na

patients

results

uptake

with

of

functional 819-826,

distal

dition

to

NCCT

expression

the

Simon

distal

et a!.

most

frequent

and

apical

other

immunoeytochemistry

thiazide-sensitive in the

polycbonab membrane

cellular NCCT

NaCI distal

eon-

antibodies of the

early

(3).

be

PBMC

used

with

from

in the

These

for GS.

domains

are

mutational Furthermore,

compound

and based

10. 1997. Accepted October 15. 1997. to Dr. Ira Kurtz. UCLA Division of Nephrology. 10833 Le Room 7-155 Factor Building. Los Angeles, CA 90095-1689.

This

populations,

estimated

0.001 and a mutant allele frequency The effect of specific mutations tional

difficult sion

properties

of the

to study, to the

renal

because distal

transporter

the

are

to the

intra-

extraceblular

or

described

thus

together

with

alleles within the are not rare. In the

prevalence

of

heterozy-

is approximately

a sporadic

1%

prevalence

of 1 in 200 (3). on the molecular and in patients

of the localization convoluted

mutations

mutations

finding,

expression

et al. have

distrib-

boss-of-fune-

patients

mutant alleles the

for

mutations

with

than

independent that mutant

phenotypic

of

localized

of the

heterozygotes.

Italian on

is evidence

Missense

rather

Many

late

(13).

frequently

protein

domains.

the

( 1 2). In ad-

loss-of-function

(6,14,15).

more

of the

there

consistent

potential

of

tubule

variety

gene

reported are

staining

cells

a wide

the documentation of same kindred, suggests Swedish

intense

connecting tubule,

NCCT

Additional

transmembrane

far

less

convoluted

the

(3, 16, 1 7). Simon

1046-6673/0905-08 19$03.00/0 Journal of the American Society of Nephrology Copyright © 1998 by the American Society of Nephrobogy

can

the

identified

been

as TSC)

GS

chlorothiazide

9%.

in osteoblast-bike

recently

(3).

the

PBMC

that the underlying cause of GS (J Am Soc Nephrol 9:

with

within

alleles

that

in with

cotransport.

tubule,

terminating

have

penetranee

indicate

measured

1998)

convoluted

gotes Received July Correspondence Conte Avenue,

NaC1

To

chlo-

to chlorothiazide.

in patients

is provided

NCCT

a

latter

findings,

PBMC,

to respond PBMC

3. The

the patients

approximately

mRNA

evidence

is defective

by that

NCCT

had

24, GGT

chromosomes.

was and

In control

GS failed

demonstrate

analysis

in intron

of these

uptake

of bumetanide.

tion

In the rat kidney, label

the parents,

portion

hypocalciuria;

by

normal

22Na

with

data,

known

by hymagnesium

confirmed

site

300

from

throughout

been

splice

significance

subjects,

DNA

in intron

codon

and abdosterone levels complete linkage be-

99%

in

functional

control

genomic

site mutation

cryptic present

uted

membrane

also

not

cotransporter (NCCT) 16ql3, and specified GS as

and

functional

and

alkalosis;

renin reported

the thiazide-sensitive on chromosome

(NCCT, rTSC

plasma

stop

patient’s

rothiazide-inhibitable

two

from the insertion

characterized

potassium

metabolic

elevated Simon

have

a premature

is a disorder renal

derived 1 19-bp

of a nearby was

California.

and a new 3’ splice site mutation in (paternal allele), which resulted in the

CAA

mutation

the

The

allele),

D. JO,t

Veterans

Angeles,

5’ splice

activation

dif-

eDNA

187).

Gil’ (maternal intron 3, CAG -

the

normal

Two

acid

Los

described

-

se-

The

amino

determine

analysis

eDNA.

920). eDNA an additional

4, generating

recessive

These

vobuted

NCCT

in the

acid had

chloride-resistant BP; and Recently,

autosomal

gene

brothers

amplified

renal

detected

amino allele

Gitelman’s

an

eDNA

be demon-

Northern in

and

heterozygote). In eDNA derived from the patient’s allele, exon 24 was deleted, resulting in a premature

between

tween locus

two

blood

mRNA

could

is expressed

to human

stop codon (after patient’s paternal

normal (I ,2).

from subjects.

NCCT

identical

peripheral

cotransport

control

This

Sepulveda

of Medicine,

previously

OAK

KURTZ*

of Nephrologv,

School

(after

(GS).

reported.

NCCT

isolated

mRNA

full-length

been

whether

NaCl

were

syndrome

have

express

healthy

linkage

cotrans-

UYEMURA, and IRA

tDivision

UCL4

chloride

Gitelman’s

to determine

PBMC

parents,

and

Sciences,

complete

sodium

mutations

Health

of Dermatology,

demonstrated

(PBMC)

defective

strated

has

for

Dii’isio,i

thiazide-sensitive

genomic

whether

and

analysis

the human

Center

LEE,*

tubule.

with

of fune-

GS has been

of NCCT In this

expresstudy,

we

820

Journal

demonstrate

of the American

that

NCCT

blood mononuclear trob subjects and

two

sis,

used

PBMC

mRNA

were

mRNA

control

the

and

Clinical

obtainable

results

and

source

from

in patients

of GS

with

BP values.

with clinical features of a noneonsanguineous

The propositus

with

nesemia.

a history

His medical

cramps 47-yr-old

brother

third

brother

There

no history

not taking

had

values

except

have

Pco,; 0.7 mg/dl ereatinine; total calcium; 3.2 mg/dl per

pg/mI

parathyroid

chemistries

and 4.3

and and a

abnormalities.

or vomiting. serum

sodium;

28 mEq/L total CO,; 10 gas, pH 7.47; 38 mmHg ionized

pg/mb

calcium;

pg/mI

1,25

vitamin 67.4

screen was laboratory

2.4-

mg/db

actin

16.6

aldosterone;

13

D.

potassium;

magnesium;

A diuretic very similar

9.2

magnesium;

supine

42 mEqfL

mg/dl

and 81 mg/dl ereatinine. brother with GS had

Isolation

42

patient’s

parents

abuse

1.1 mg/dl

168

sodium;

calcium;

Both

-ii,

1.4

abuse

The

136 mEq/L

mmobIL

renin;

64 mEq/L

1 mg/dl

phorus; 47-yr-old

plasma hormone;

were:

chloride;

Urine

58 mEqfL mg/dl

1.4-

phos-

negative. values.

The

Kidney

PBMC

of PBMC

PBMC and

h supine

1.25

phosphorus:

2.4-

presented

for occasional

no electrolyte

were:

1).

hypokalemia

cramps.

2.4 mEqfL potassium; 94 mEqIL chloride; mg/dl blood urea nitrogen: venous blood

ng/ml

NCCT

4.4-

had

hypomag-

or diuretic

of chronic

of laxative/diuretic

in the propositus

who and

medications.

muscle and

history

man

of laxative any

a history

are asymptomatie

of GS (Figure marriage

hypokalemia

unremarkable

and intermittent

was no previous

Laboratory

was

he was also

hypomagnesemia,

se-

and magnesium hypocalciuria, and

a 42-yr-old

chronic

He had

and

was

of

history

in his calves.

or of vomiting,

DNA

GS.

hypokalemia, hypomagnesemia, renal potassium wasting, chloride-resistant metabolic alkalosis, UCLA

7.5-

of NCCT

genomie

Kindred

A kindred was identified Two of the three offspring

to

peripheral

Methods

Features

normal

in

studies were performed to determine be used to assess NCCT function in

subjects

Materials

is expressed

as an easily

Additional PBMC could

healthy

of Nephrobogy

cells (PBMC) obtained from healthy conbrothers with GS. For mutational analy-

to complement

quencing. whether

Society

from

were

isolated

healthy

control

from

the two patients

subjects

as follows:

with GS. the parents, 15 ml of heparanized

Figure porter

2. Northern blot of thiazide-sensitive sodium chloride cotrans(NCCT) mRNA. Kidney and peripheral blood mononuclear

cells (PBMC) poly(A) RNA was probe and then rehybridized with poly(Ai

H

blood

a

RNA

was added

method ison,

affected

1. Gitelman’s siblings.

syndrome

(GS)

Filled

symbols

added.

RT 6000B

cell

layer

was isolated

saline

cells

were

was

to which

generated

I 2 ml

centrifuged

centrifuge,

at 2100

and the supernatant

resuspended

from PBMC and Sacehi

with the PolyATract WI).

specific indicate

The

in RPMI

1640

+

centrifuged in RPMI

a

Analysis RNA

The

mRNA separated

RNA

was

I .7-kb

PCR

product

with

the

following

2.2 M formaldehyde

49 kindred.

The

ofChomezynski

PBMC,

Figure

was then

discarded.

Total

47

an NCCT-speeifie Loading: 1 p.g of

glutamine + streptomycin/penicillin. The cells were then at 1600 rpm for 20 mm, and the pellet was resuspended second time.

Northern

42

to 25 ml of phosphate-buffered

rpm for 20 mm in a Sorvall

]

with probe.

per lane.

ofFicoll-Hypaque

was

hybridized an aetin

CCACTGATC.

(18).

and

(antisense)

Poly(A)’

kidney was

using

isolated

the from

isolation system (Promega, Madon a 1 .2% agarose gel containing

and transferred (from

and human

to a nylon human

primers:

membrane.

PBMC (sense)

NCCT

An NCCTeDNA)

GACCAGCTGTAC-

GAGCTGTGGACAGGGATGTC.

was

PBMC

a.

V

C.

Figure and

3. Genomic

patient

had

in intron

DNA

sequence

ANTCCTGACCTGG

of intron

site mutation

3/exon

4 boundary:

in intron

3, CAG

(a) Father;

CAA,

-

was random-primed

of approximately 42#{176}C for

with [32P1 dCTP

I .5 X lO

2 h, using

50%

dpm/j.tg.

The

formamide,

ethylenediaminetetra-acetic

acid,

6X

0.5%

to a specific

filter

was

saline-sodium

sodium

activity

prehybridized

dodecyl

phosphatesulfate

probed at 42#{176}C for 18 h and washed in I X SSC, 0.1% for 60 mm (3 changes, 350 ml per wash); after exposure

filter was rewashed DNA

activity

T4

PNK

labeled

.tCi/pmol.

The

formamide,

minetetra-acetie sheared

in 0.1 X SSC, 0.1%

was

of 2.5 50%

(SDS),

acid,

herrings

0.5%

SDS at 65#{176}C for 60 mm. The

with

filter

6X

testes

in 1 X SSC,

Analysis

[32P]

y ATP

was prehybridized

saline-sodium SDS,

DNA.

to a specific at 42#{176}C for 2 h,

phosphate-ethylenedia-

Denhardt’s

denatured

0.1%

ofNCCT

SDS

DNA was and healthy control kit

unaffected using

primers

sequenced

Total

821

GACCTGG

with

GS;

remaining

(d) Healthy

allele

had

total

RNA

control.

the consensus

solution, After

the

(Invitrogen).

sibling.

were

All 26 exons an ABI

RNA from PBMC

and

unable

and 0.1 mg/ml prehybridization,

cDNA

of the NCCT

by Simon 310

et a!. (3).

automated

was obtained

gene

The

DNA

were

genomic

sequencer

from both patients

reverse

was

Both

the father

CAG

sequence

reverse-transcribed

transcriptase.

alleles

and maternal

Results).

In the region

the following with GS and

revealed

of the paternal

primers healthy

two

allele

with

Full-length

the paternal

control eDNA

different

mutation

NCCT

subjects derived

was from

mutations

(see

(exons

3 and 4),

were used to amplify eDNA from both patients control subjects: (sense) CAGGCGACAATG-

GCCATTG. In the region of the maternal allele mutation (exons 23 to 25), the following primers were used to amplify eDNA from both patients

with

(Perkin with

from

the

and

healthy DNA

(300

malities

in the patients.

healthy

was in

a

1 CaCb.,,

of the cell

suspension

12-X-75-mm

water NaK2C1

Elmer).

100

GS, and

New

pA

for

of solution

(n

=

solution

containing

I MgC12,

5 Hepes,

10 mm

tubes

with Isotope

A containing Nuclear,

in 150 with

from

6).

CGTGTGThealthy

control

the sequence

abnor-

Boston,

the patients,

PBMC (in

mM):

d

bumetanide uptake 22Na MA)

initiated

(5 .tCi/ml, with

and

NaCI,

2.5

A). Aliquots were

placed

in

at 37#{176}C in a shaking

(0. 1 mM)

was

washed

140

per tube)

and preineubated

the parents,

were

pH 7.4 (solution

(3 X 106 eells/l00

cotransporter. England

sequence compared

in PBMC

subjects

polystyrene

bath

(sense)

Function

measured

control

K,HPO4,

amplified

was

of NCCT uptake

subjects:

AGGGTCTCCAGCCAGGC-

NCCT

chromosomes)

Assessment

control

(antisense)

subjects

DNA

was

GS

TCGTAGGCGGCCAG, CATG. The genomie

resuspended

both patients with Gen DNA purifi-

to obtain

The virus

with GS and healthy In both patients, NCCT

22Na

isolated from the parents, subjects, using the Turbo We

described using

DNA

subjects.

myebobbastosis

eDNA from both patients amplified and sequenced.

at 45#{176}C for 30 mm.

Genomic

Genomic cation

The

control

avian

and

GS,

mRNA

SDS at 45#{176}C GCAGAACT, (antisense) CCAATGATGCGGATGTCGTF; nested, for 6.5 h, the (sense) GTGCGCTFCGGCTGGGTC, (antisense) GACTFGACC’IT-

the filter was probed with the 32P probe, using 25 ml of hybe buffer. The probe was denatured and added to the hybe solution at 0.5 pmol/ml. The filter was probed at 42#{176}C for 18 h and then washed 3 times

(e) Patient

allele.

healthy at

Denhardt’s solution, and 0. 1 mg/mb sheared herrings testes denatured DNA. After prehybndization, the filters were incubated with the 32P probe, using 25 ml of hybridization buffer. The probe was denatured and added to the hybridization solution at l0 dpmlml. The filter was

using

NCCT

3.

The probe

aetin

one

AGTCCT

C

(b) Mother;

affecting

Mutated

C AGTCCTGACCTGG 10

d.

V

C

a 3’ splice

b.

GACCTGG

10 C ANTCCT

Express

to inhibit

the

at 37#{176}C by adding carrier-free,

bumetanide

Dupont (0. 1 mM).

822

Journal

of the American

Society

AACAGAGTCAAGGGC

V

C.

AACA

Figure

4. Genomic

DNA

sequence

mother

and

had

a 5’ splice

sequence

in intron

Tracer and

uptake

after taming acid

linear

AA

G N T G C

24/intron

mutation

24 boundary:

and was

measured

22Na

of ice-cold

in the presence

uptake

isosniotie

140 tetramethybammonium

was stop

pH 7.4 (solution 4#{176}C (GS-6 refrigerated CA). After an additional in a heating

centrifuge,

cycle I ml of 0.2N

block

for

Coomassie

brilliant

(19).

Beckman

Instruments.

of pelleting and washing, NaOH to each tube and Aliquots

fluid

(Ultima-Gold.

blue

Separate after

boiling

con-

140 gluconic

for

G250

samples

adding

5 mm)

of cell

lysate

Packard.

with

bovine

serum

of cells

(which

underwent

I ml of

I 2%

used

as blanks

were

Fullerton,

anide

(Sigma,

(Promega); inger cataway.

were

used

St. Louis, avian

Mannheim, NJ),

A AC

-

with

allele.

one

The

myebobbastosis Indianapolis.

Turbo

Gen

virus IN):

derived insertion

as the stanuptake

per tube from

and

the total

DNA

reverse

purification

and bumet-

isolation

system

transcriptase

kit (Invitrogen,

propositus

the

In

addition,

Both

consensus

the GGT

5’

splice

allele)

patient’s

site (3).

eDNA

from

the

mature

stop

pletely

sequenced,

I 2 1 bp

prior

site

was

the

1 l9-bp

codon

derived resulting

acid

and

a cryptic

upstream

920

Pis-

Functional

NCCT

the 2, human transcript.

PBMC Full-length

express NCCT

splice

patient’s

from

site

of

6. eDNA 1 1 9-bp pre-

3 was

com-

was

found

CAG

4 boundary. allele,

prior

This

resulting

to exon

maternal

in a predicted

(Figure

Intron

paternal

the patient’s

and

a predicted

3/exon

DNA

OTT

analysis

additional

I 87).

intron

GOT

5

an

mutation

allele,

premature

stop

in

4 (Figure exon

codon

24 after

6).

Analysis

Additional

Carlsbad,

Analysis in Figure

had

of 3 and

a previously

mutational

4, generating

of intronie

was

amino

in Figures

acid

site

24,

shown allele

sequence

and

intron

are

in the

6). In eDNA deleted,

in

of the

results

in Figures

3’ splice

results

amino

the

allele),

mutation

(after

insertion

a new

was

The

because

The

3 and

to the

activated

had

exons

siblings

As shown

(paternal

paternal

between

shown

DNA

genomic

affected sequenced.

identical.

CAA

-

two

were

are was

genomic

3. CAG

the

exons

experiments

function

were

was abnormal

eotransporter

function

ehborothiazide-inhibitable

4.5-kb

control.

had

(Boehr-

(Pharmacia,

Results shown

(d) Healthy allele

sequenced.

and

NCCT

siblings

patient’s

using

the same

Ficoll-Hypaque

from

4, the

NCCT

Mutational

GS;

other

was

parents

all

affected

( I 600-

acid

mRNA

obtained

both

siblings

both

and

counting

chborothiazide

PolyATract

isolated,

for protein

and subtracted

in this study:

MO):

affected from

the

albumin

trichloroacetic

both DNA

NaCI

mately

A A 0 G T G C

(e) Patient

affecting

Grove,

Downer’s

CA)

As

A G A GTC

(b) Mother;

GTT.

in intron

uptake.

Materials The following

U.

the cells were heated at 75#{176}C described (maternal were transferred

IL), and the radioactivity was quantified by scintillation TR, Packard). The remaining cell lysates were assayed

procedure

solution

hydroxide.

10 mm.

to 10 ml of scintillation

dard

terminated

7 Ca gluconate. 2 Mg gluconate. 5 Hepes. B). The cells were pebleted at 2400 rpm for 5 mm at

by adding

AACAGAGTCAAGNTGC

(a) Father;

24. GGT

in intron

2.5 K2HPO4.

lactone,

lysed

site

( I mM).

by the addition

(in mM):

TC

of exon

for 2 mm

of chborothiazide

1 mm

G AG

U.

24.

was

absence

V

1

a.

patient

Nephrobogy

of

patients

an approxi-

healthy

eDNA

zide

from

with

control significantly

done

to

was

assayed

22Na uptake OS,

each

subjects. decreased

parent As

determine

whether

in the two brothers

shown 22Na

OS.

The

by

measuring

the

in PBMC

obtained

from

carriers),

and

(heterozygote in

with

Figure

uptake

7B, in

chborothiaPBMC

from

PBMC

A

5. (A)

PCR

GCAGAACT, CATTG.

were

exon

amplification

detected

4. (B)

2 of NCCT

3

eDNA

from

(antisense) CCAATGATGCGGATGTCGTT; I , 1-kb ladder; lane 2, normal PBMC;

Lane

(lower)

PCR

in the patient

amplification

with

GS.

of NCCT

The

eDNA

lane

from

(antisense)

AGGGTCTCCAGCCAGGCCATG.

control

two

407

a deletion

bands,

of exon

healthy

control

PBMC

from

24.

Both

bp (upper)

patients

subjects

and

the patients

with

the

with

(exons

(sense)

3, patient

with

PBMC

(exons

bp (bower),

GS

had

identical

but

had

et al.

NCCT

OS

detected

effect

(3).

The

firmed

by

other

mutations

found

to be

with

distributed

more

intracellular

rather

than

of the

patients

In the

permitted mRNA of

tional

analysis

intron

patients

maternal

we

far

have

The This

obtaining 24

thus

on

using

exon

24

of

at the allele

mRNA

823

domains

was

that analysis

especially tissue

mRNA

level.

reported was

shown

analyzed

from The by

NCCT

PBMC

protein

also

at

given

the

patients

5’ splice Simon

to result

et

genomie

the site

in the

muta-

mutation (3) deletion

in

(20).

affected

patients.

The

functional

PBMC

isolated

protein. studied.

demonstrated

(unpublished

NKCC2

study

mRNA.

may

be

equally

the of

results

to generate of these

the

patients

had

We

have

uptake.

the

that

PBMC

methodology

useful

in patients

in the

present

we

with

used Bartter’s

(21). patients

effect

of a given

plete

loss

mRNA

insertion

The

observations)

Therefore,

in

Therefore,

consequence 22Na

and 4

subsequent

be predicted from

B.

site

i.e.,

deletion would

Unlike

exon

on

certainty,

characterized

the was

ent loss-of-function mutations. It would PBMC from compound heterozygous

imprac-

for

The

two

of

mutation

to

mutations

mutations

pound heterozygotes, and therefore thiazide-sensitive NaCl cotransport

NCCT

the

al.

is

allele.

in the

both

site

the paternal with

bp

revealed

splice

prior

exon

GS. A and

splice cryptic

thiazide-inhibitable

express

syndrome

mRNA

both

recently

3’

site

201

band

in Panels

fragment

predicted

versus

with

of the lower

new

splice

be

demonstrable

in this

Many

express

useful

was

A

genomie

eDNA

NCCT

3, patient

of an upstream

from

that

lane

and

to the beginning

CGTGTGTI’CGTAG-

are depicted

derived

NCCT

mutations no

patient

bp (upper)

prior

sequence

intronic

sequence

truncated

been reand are

heterozygotes. NCCT

intronie

320 (sense)

PBMC;

1 l9-bp

of

bands,

mRNA.

cannot

demonstrate

Additional

domains.

that

effect editing

eon-

of the

compound

The

CAGGCGACAATG-

GACTFGACC1TOC-

insertion

The

activation a

(sense)

primers:

OS.

of one

mRNA

been

genomie

with

NCCT

insertion

of

the

in in the

RNA

two

intronie

lane 2, normal

resulted

to

spec-

eDNA,

the following

the results

pen-

of (3).

OS

primers:

(antisense)

a I l9-bp

in the patient

99%

have recently are most frequent

shown

finding

renal

previously

has

variety throughout

are

a mutational

level.

ticality in

study,

with

or transmembrane

described

in PBMC.

mRNA and

the extracellular

present

expressed

to the

and

alleles

mutations mutations

localized

wide

loss-of-function

potential loss-of-function ported (6,14,15). Missense often

of

l6q13,

16ql3

A

(4-8).

linkage

disorder

to chromosome

groups

were

chromosome

recessive

linkage

consistent

complete

on

control

revealed

therefore,

OS.

demonstrated

(SLCJ2A3)

as an autosomal

etrance

gene

first

gene

NCCT

3

the following

Unlike

NCCT

Simon ified

GS. band

I , I -kb ladder;

were findings;

no

4), using

23 to 25),

Lane

271

2

GTGCGCTTCGGCTGGGTC,

of the upper

Discussion the

3 and

nested,

and

parents,

1

PBMC

sequence

GCGGCCAG, eDNA,

Mutated

B

1 Figure

Express

of

missense function.

in heterobogous

mutation The expression

study

be more patients

that

expression systems

were

com-

absence of PBMC a result of two differ-

does of

difficult using to study the not

cause

mutated would

comNCCT

be

required

824

Journal

of the American

Society

of Nephrobogy

A Normal

GCGGCTGCCC

TGGATTACGG

CCCAGGCAGG

CATCG

1-F

GCGGCTGCCC

TGGATTACGG

CCCAGGCAGG

CATCGATGAG

2-M

C.CCCJCTGCCC

TGGATTACGG

CCCAGGCAGG

CATCG

CTCAGAGAAG

GGAGATGAAC

GTAGGTCGCA

TGGTGAATGA

505

520

AAAACTGAAC.

505

505

Normal 1-F

GTAGCJCAAAC

570

2-M

505

Normal

505

1-F

TGGGGCTCCT

CCCTTGGGAA

ATGCCCTGCC

TAAGCTTFGG

GTGCCACCCT

620

2-M

505

Normal

- - - - TCCTGA

CCTGGATCAT

CATCCTGCTG

TCGGTCACGG

TGACCTCCAT

1-F

GCAATCCTGA

CCTGGATCAT

CATCCTGCTCI

TCGGTCACGG

TGACCTCCAT

551 670

2-M

----TCCTGA

CCTGGATCAT

CATCCTGCTG

TCGGTCACGG

TGACCTCCAT

551

Normal

CCTGACATCA

ACCAGAACCC

TCGGGCTGAG

CACACCAAGA

CGTTTGAGGA

1-F

CCTGACATCA

ACCAGAACCC

TCGGGCTGAG

CACACCAAGA

CJGT’I’T(;AGGA

2-M

CCTGACATCA

ACCAGAACCC

TCGGGCTGAG

CA

Normal

CATGATTCTCA

CCCTTCCGTC

TC,AATGATGG

C11’CAACJCAT

GAGGCCACTG

1-F

CATGATIC.CA

CCCTTCCGTC

TGAATGATGG

CTFCAAGGAT

GAGGCCACTG

B 2765 2765 2747

2815

2815

2-M

2747

Normal

TCAACCAGAT

GCGGCGGGAC

TGCCCCTGGA

AGATCTCAGA

TGAGGAGATT

2865

1-F

TCAACGAGAT

GCGGCGGGAC

TGCCCCTGGA

AGATCTCA(;A

I’GAGGAGATT

2865

2-M

2747

Normal

ACGAAGAACA

GAGTCAACPTC

CCTFCGGCAG

GTGAGC.CTGA

ATGAGATTGT

2915

1-F

ACGAAGAACA

GAGTCAAGTC

CCTFCGCCAC

GTGAGGCTGA

ATGAGATTGT

2915

2-M

Figure

6. Sequence

eDNA derived maternal allele from

from (2-M)

the paternal

allele

the

However,

in patients

loss

of

protein

(1-F)

functional

function

of

targeting,

in the

T transition

gene,

12 nueleotides

was

hypothesized

in a patient

NCCT

mRNA

Northern

analysis

In heterozygote trolytes these

are

within

individuals

to

with

will and

allow

this

the

normal subtle

GTGAGGCTGA

(A)

Two

ability

range,

PBMC.

port 22Na

et al.

have

region

of

mRNA PBMC

addressed

(23). serum might salt

and

using

urine

elec-

speculate and

potassium

that

allele

with in

in

from

PBMC

Previous

studies renal

bow

bevels

The

results

expression

in

have

calcium

excretion.

the

whether

NaCl

two

in each

24.

to determine

PBMC of

(10),

cotrans-

thiazide-inhibitable patients

was

NCCT parent

not

de-

mRNA/protein accounts

that prostate,

study NCCT

was

in PBMC.

NCCT

for

is expressed

osteoblast-like

intestine,

NCCT

urinary

the

shown

of the present

transport

of exon

these

study.

of full-length

Furthermore,

of

allele

cortex

small

a deletion

measured

further

in the patient.

of the eDNA from the sequence of the eDNA

thiazide-sensitive

parents

normal

requires

mammalian

had

upregulation

the

detected

be of interest

The

the

Whether

derived

were

decreased

also

can be detected.

findings

(2-M)

it would

uptake

2779

sequences

of exon 4. The sequence detected in the patient. The

differences

creased.

sodium

eDNA

associated

subtle

binding.

NCCT

the one

In addition,

to measure

PCR

detectable

wasting

ATGAGATTGT

different

the maternal

(22).

eodon,

to be

from

fune-

the start

steady-state

although

in

thiazide

from

derived

complete

5’ untranslated

issue

but

cause

Mastroianni

The

quantitative

carriers, have

alter

(14).

the

studied

upstream

OS

mutations

not

affect

stability.

a C -

levels

did

potentially

mRNA

the NCCT which

that

be

eDNA

mutations,

potentially

described

CCTTCGGCAG

to GenBank).

region.

of these

a mutation also

according

in this

homozygous

could

and

normal

consequences

could

in NCCT

(numbered

allele ( I -F) had a I 19-bp insertion prior to the beginning in this region. (B) Two different eDNA sequences were

was

with

consequence

Mutations

eDNA

the paternal was normal

to address tional

TC

of NCCT

cells colon,

provide

the

in human

shown The

this

first

nonepithelial

to mediate role

and

in the

(13),

and

(7).

evidence

for tissues.

a component transporter

at

spleen

plays

of in

PBMC

A

Gitelman

HI,

Graham

characterized soc Am 2.

22Na Uptake

DB,

Bartter’s

20

40

60

100

80

120

140

Time ( sec)

in the

Pollak

J Ant

10

6.

( ‘k Change)

7.