Kl£u HiNH COA BENH CU'ONG INSULIN BAM SINH

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TAP CHl NGHIEN CU'U Y HQC

Dl TRUYEN PHAN TU, TUONG QUAN KI^U GEN - Kl£u HiNH COA B E N H C U ' O N G INSULIN BAM SINH

Dang A n h Du'O'ng, V u Chi D u n g , Cdn Thj Bich Ngoc, N g u y i n Phu Dat, Trdn Minh Dien Bdnh vidn Nhi Tmng wong Nghidn cOv nhim xic djnh dpt biin cie gen ABCC8. KCNJ11, HNF4A va GLUD. twang quan giira kiiu gen - kieu hinh cua cdc bdnh nhan cwdng insulin bam sinh Ket qua phat hidn thay ed dot bien gen 43.8%

trwdng hap, trong dd dpt biin ABCC8 (37,6%), KCNJ11 (3.1%), HNF4A (3,1%) 100% cac trwdng hap cd hai dgt biin lin ho$e mdt dgt bien trpi tir bo cua gen A8CC8 khdng dip irng vdi diazoxide va phai phiu thudt eit tuy 95%), cac trudng hap khdng cd dgt bien gen thwdng dap irng vdi diiu tri ngi khoa. Tre cd cwdng insu- lin bam sinh can phan tich dgt bien gen de giup cho chan doan va quyit dinh dieu tn. NhOng gia dinh cd tre bl cwdng insulin bim sinh cin tu vin di tmyin, chin doan trudc sinh va theo doi didu tri ngay sau sinh.

Jir khoa: cwi/ng insulin, d^t b i l n gen ABCC8, KCNJ11, HNF4A, GLUD

I. DAT

V A N

Dt

Cudng insulin bam smh Ici m6t benh vd'i bieu hi?n hg glucose mSu n^ng k6o d^i o tre so sinh v& tre nho do r6i logn di^u h6a bSi ti^t insulin. ChSn doSn sb-m Vci dieu tri kip thd'i, kh6ng tri ho§n 1^ v6 eCing quan trpng nhSm han eh^ t6n thuong nao v^ di chung vTnh vi§n than kmh eho tre. Nguyen nhan ph6 bi^n nhat vS n^ng nh^t eOa b0nh di truyen nSy lien quan d i n cSc dot b i l n l$n bat hoat k&nh KATP O"

mSng te b^o p eua t i i u dao tuy. Kenh KATP dieu h6a quS trinh khoi ph^t eho vi0e bat tiet insulin. CSc dot bien Ign gSy b6nh cud'ng insulin bam sinh the hien o- m5t trong hai don vj cau triie nSn k6nh KATP '^ sulfonylurea receptor 1 ( S U R I ) hoSe l5 dieu hoa ion cua n6 Id Kir6. 2 Hai don v! protein n^y dup'c m§ h6a bdi eSc gen n^m cgnh nhau tren nhiem s i c

Bia Chilian h$- 0$ng Anh Duong - Khoa H6I SI bdnh vi$n Nhi Tmng uong

Email: danganhduong.sicu@yahoo com vn Ngdy nh$n-13/1/2014

Ngdy duoc chip thudn 29/8/2014

t h e n l d / \ e C C 8 v S ; < C A / J 1 H 1 l - C a c d O t b i e n I3n xuSt hi?n d ea hai alen (tCr bo v^ me) gSy nSn t§ng sinh lan toa te bdo p cua tieu dao tuy. The tSng sinh cue b6 cua t l bdo |3 thudng xuat hien khi e6 dot b i l n trpi tao kenh KATP nguon goc tir b6. Dieu trj can thuc hien ngay b l n g cung eSp glucose toe 66 eao (glucose tTnh mach hoac glucose da day), si> dyng diazoxid hoSe octreotid, can thi6p p h l u thudt cat bo 95 - 98% tuy n i u d i i u tri n6i khoa that bai [2, 3]

Ct Viet Nam, cudng insulin bam sinh cung Id b&nh canh thudng gap trong ciip eiru va hoi sCre. Trong giai doan t u 1/1/2010 den 31/8/2012 nhdm nghien edu gdp 32 trudng hp'p mdc benh. De tai ndy dupe tien hdnh nhdm muc tieu sau;

Xac dinh dpt bien cdc gen ABCC8, KCNJ11, HNF4A vd GLUD1 tren cae bSnh nhdn d u o c chan dodn cudng insulin b i m sinh.

Nhan x6t t u o n g quan giua kieu gen - kieu hinh cua cdc benh nhdn cudng insulin bam smh

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TAP CH! N G H I N CI>U rrtpc'^

II. D 6 | TiraNG VA PHU'aNG PHAP

1. Ddi tu'O'ng

32 b0nh nhdn c u d n g insulin bam smh dup'c c h i n dodn vd dieu tri tgi benh vien Nhi Trung uong t d 1/1/2010 den 31/8/2012.

2. Phivvng phap

2.1. Thiet ke nghidn CLFU: nghiSn cCru tien eCru mo ta cdt ngang, khdng e6 nh6m ehCrng.

2.2. PhuKJiig ph^p Tieu chuin li/a chon:

Ti6u chuan chan dodn eua Hussain K.

(2008) [4].

Bfinh nhdn dup'c chan dodn cudng insulin bam smh khi eo du ede ti§u chuin sau:

Glucose mau ha lue ddi hodc sau khi Sn (<

2,5 - 3 mmol/l). K i t hp'p vdi tdng t i l t insulin vd c-peptid (insulin h u y i t thanh > 1 mU/l)

Gdp ung vdi tidm glucagon, glucose mdu tdng 2 - 3 mmol/l sau ti6m d u d i da 0,5 mg glucagon,

Khflng e6 ceton ni&u vd xeton mdu thdp.

Toe dp truyen tTnh mgeh glucose de duy tri nong 66 glucose mdu binh thudng (> 3,5 mmol/l) Id > 8 mg/kg/phut.

Khdng c6 ede b l n g chung eua ha glucose mdu do cdc roi loan chuyen hda bam sinh {acid beo, acid h&u c o , acid amin).

Tieu chuin lo^i trir

Cdc tre so sinh ha glucose mdu do cdc nguydn nhdn khde nhu: me m I e ddi thdo dudng, chdm phdt trien trong t u cung, nggt, ede roi logn chuyen hda bam sinh, ede b§nh di

truyen khde (trisomia 13. hdi ehdng Turner, ,.,), toe dd duy tri t r u y i n tTnh mgeh < 8 mg/kg/

phut d l e6 n6ng d6 glucose mau binh thudng.

Ph^n tich gen

Cdc tre dug-c chan dodn c u d n g insulin bam sinh theo tieu c h u i n tren se dup'c liy mdu chiet tdch DNA theo quy trinh chuan tir bach cau lympho mdu ngoai vi cua b^nh nhan vd b6 me. Exon d o n ddc eua gen KCNJ11 vd 39 exon eua gen ^ S C C 8 dup'c khuyich dgi b l n g ky thudt PCR vd giai trinh t y thee quy trinh cua Ellard S. vd Flanagan S. E [5, 6], Phan Crng giai trinh t y dwac phdn tieh trfin ABI 3730 capillary sequencer {Applied Biosytems, Warrington, UK) vd dup'c so sanh vdi trinh tie gen dd dup'c cdng bo (NM_000525 and NM_000352.2) sCr dung "Mutation Surveyor version 3.24" (Softgeneties PA, USA).

Dao diFC n g h i § n cu>u

Nghidn cCru ndy d u o c s u dong y cCia b6 me b§nh nhdn. Bo, me cua benh nhan duoc thong bdo day du ve myc dich nghien cuu vd cd q u y i n tut lui khdi nghien cuu b i t ky luc ndo.

III. K t T QUA

I. Di t r u y e n p h d n t u '

Ty 1$ gap dpt bien gen ABCC8 hay g^p n h i t chiem 37,6% (bang 1).

Trpng so cdc b$nh nhdn e6 dpt b i l n gen ABCC8 thi cd phdt hien d u o c mdt d^t bien mdi d vi tri exon 15 khi d6 c 2056T > A, exon 34 khi do e. 4135G > A vd exon 8 khi d6 c, 1183A > T. Ddy la cdc ddt b i l n ndy chua tCrng d u o c cdng bo trong ban do ddt bien gen cua gen ndy (bang 2)

TCNCYH 89 (4) - 2014

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• TAP CHi NGHIEN CCPUgf Hpc Bang 1. Loai dpt bien gen trgn 32 benh nhan cifd-ng insulin bam sinh

Gen

ABCC8 KCNJ 11 HNF4A GLUD1 T6ng

Benh nhdn bj d$t b i l n n

12 1 1 0 14

%

3 7 . 6 3.1 3,1 0 43,8

Bang 2. Cac Itilu dpt b i l n ABCC8 tren benh nhan cu>6>ng insulin bam sinh

B|nh nhan

1

2

3

4

5

6

Dpt biln alen 1 (c. DNA)

c. 2056T > A

c 2057T > C (exon 15) c 3403-1G>

A (acceptor site intron 27}

c. 2057 T > C c 1467+5G>

A (intron 9) c. 3 4 0 3 - 1 G >

A (acceptor site intron 27)

Dpt biln alen 2 (c. DNA)

c. 2057T > C c 3403-1G>

A (acceptor site intron 27) c. 3403-1G>

A (acceptor site intron 27}

c. 2995C > T c. 2800C > T (exon 23) c. 3 4 0 3 - 1 0 >

Dpt biln alen 1 (protein)

p F686I

p.F686S

Aberrant splicing

p. F686S Aberrant splicing Aberrant splicing

Dpt biln alen 2 (protein)

p. F868S

Aberrant splicing

p. R999X

p. R934X

Aberrant splicing

Dpt biln ti>bd

p F686I

p. F686S

C.3403-1G>

A (acceptor site intron 27) p. F686S Aberrant splicing c. 3 4 0 3 - 1 0 >

Dpt biln tirmq

p. F868S c. 3403 - 1 G >

A (acceptor site intron 27) c, 3403-1G>

A (acceptor site intron 27) p. R999X

p. R934X c. 3403-1G>

2. Tuxrng quan kilu hinh va kilu gen

Trong nghifin cdu ddp Crng vdi dieu trj Diazoxid, thi 100 % cdc ca khong e6 dot biln cdc gen ABCC8; KCNJ11, HNF4A vd GLUD1 Id ddp Crng vdi dieu tn (bang 4).

Trong cdc ca cd hai dOt bien ldn hodc mdt dot biln trOi tir bo thi 100 % Id kh6ng ddp Crng vdi diiu trj ndi khoa vd can dup'c can thiSp blng phdu thu$t cdt tyy 95%, Neu ed mdt dOt bien tr6l tir me thi ed ddp Crng vdi dilu tri npi khoa (bang 5).

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TAP CHI NGHIEN CU'U Y HQC

Bang 3. Cac kilu ddt biln ABCC8 tren b^nh nhan cuidng insulin bim sinh Benli

nhan 7 8 9 10 11 12

Dot biln alen 1 (c. DNA)

c . 3 4 0 3 - 1 G > A {acceptor site intron 27

c. 3 4 0 3 - 1 O A (acceptor site intron 27

c. 4135G > A (exon 34)' c 1183A>T

(exon 8)*

c. 4160_4162del (exon 34) c. 2057T > C

(exon 15)

Dot bl§n alen 2 (c. DNA)

ND ND ND ND ND ND

Dpt biln alen 1 (protein) Aberrant splicing Aberrant splicing p. G1379S

p. I396F

p. S1387del

p. F868S

Dpt bign tCrba

c. 3 4 0 3 - 1 O A (acceptor site intron 27

c. 3 4 0 3 - 1 G > A (acceptor site intron 27

p. G1379S ND p. S1387del

p. F868S

Ddt bi§n tif me

ND

p i395F

ND

* DOf biin mOI.

Bang 4. Tu'O'ng quan kilu dot biln gene vd'i dap ipng vai diazoxid Kieu gen Ki4u hinh - dap ifng vd'i diazoxid Khong dot bien ASCC8; KCNJ11;

HNF4A vi GLUD1 **' Sdca

18

Opt bi6n HNF4A (+) vi ngirng thuoc 1 06t bien KCNJ11 (+) vi con ha giucosemSu nhe 1

Bing 5. Tirong quan l<ilu hinh vi I<i4u gen cua cic ca d6t bijn ABCC8 _., Kieu hinh - dap frng

C6(n) Hai aot bien iSn ASCC8

Mat aoi bien trSi ABCC8 tCr b6

Mot an bien troi ABCCS tir me 1(1)' voi diazoxid

Khdng (n) 6 (6)"

5(5)"

* Khong cdn cSt tuy; ** Clf 95% ttty n0i soi

TCNCYH 89 (4)-2014

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• TAP CHi NGHIEN CO'U Y Hpc IV. BAN LUAN

Xdt nghidm ve di t r u y i n phdn t u trong t h y c hanh Idm sdng ehSn dodn, d i l u trj, t u v l n di truyin vd chan dodn trudc sinh doi vdi hp glucose mdu ndng do c u d n g Insulin Id mdt trong nhO'ng thdnh t y u cOa y hpc hi^n dai. & Vidt Nam, l l n dau tidn chCing t6i t r i l n khai vi$c Crng dyng phdn tfch dOt b i l n gen trong thyc hanh Idm sang chan dodn, d i l u tn vd t u van di truyen d i i vdi ha glucose mdu n§ng do cudng insulin b i m sinh tCr ndm 2010, Viec Crng dyng ndy rd rdng Id ea s d khoa hpe tin edy trong vi$c lya chpn ehi djnh dieu tri thich hdP, dac bi^t trong trudng hp'p phai ph^u thu^t cdt gan todn bd tyy. Cho d i n nay 8 gen da dup'c b i i t d i n c6 liSn quan d i n cudng insulin b i m sinh e6 tinh c h i t gia dinh. Dot b i l n gen ABCC8 Id nguySn nhdn chinh cua benh va dup'c md ta dau ti&n, khoang 45% cdc trudng hp'p cudng insulin b i m sinh ed ddt bien d gen ABCC8 vd khoang 5% cdc trudng hp'p ed dot bien d gen KCNJ11 (Cdc protein dup'c md h6a bdi ABCC8 vd KCNJ11 tham gia c i u tgp nen kdnh K~ATP cua t l bdo p t i i u dao tyy vd co ehde ndng d i l u hda bdi t i l t insulin. Khoang 5% bdnh nhdn e6 dOt bien a gen GLUD1. day la gen md hda eho enzym glutamme dehydro- genase (GDH) H i i m han Id cdc ca c6 dot b i l n d gen GCK. Id gen ma h6a cho enzym glueokinase, ddt b i l n gen UCP2 vd dOt bien ldn cua gen HADH. Cd khoang 40% b$nh nhan khflng tim thay dot b i l n a cae gen. Hien vdn chua rd Id nh&ng bdnh nhdn khflng phdt hi$n dup'c dOt b i l n e6 t h i e6 dot b i l n intron, hodc cdc dot b i l n mat dogn Idn (thudng khdng phdt hien dup'c bdng cdc ky thu^t dang dp dyng hi$n nay cho cdc gen nay} ho|ie ed ede dSt bien d cdc gen khde md chua xac dinh dup'c [7] Trong nghidn cCru cue ehOng tdi phdt hi^n d u d c 14/32 (43,8%) e6 d6t b i l n gen (trong dd d^t b i l n ABCC8 (37,6%), KCNJ11

(3,1%), HNF4A (3,1%) vd khSng trudng hap ndo d u a e phdt hi§n ed ddt bien gen GLUD1, GCK, hpdc HADH). Nhu vdy, nhin chung nh6m b§nh nhdn cua chung toi eo ty le phdt hipn gen t u a n g t y nhu bdo cdo eua James d tren. trong dd dpt bien gen ABCC8 Id hay gdp nhdt (37,6%} cdc trudng hp'p ed ddt b i l n gen.

Nhung so vdi nghidn eCru eua tde gia K. E.

Snider, phdn tich gen 417 trudng hap cudng insulin bam smh, t^ le phdt hipn dot b i l n gen eua tdc gia ndy cao han nhieu (79%), [8].

Trong nhdm bdnh nhdn ddt b i l n gen ABCC8 dup'c phdt hien, xdc dinh dyp'c 9 dpt b i l n khde nhau trong 66 cd 3 d6t b i l n (c 2056T > A (exon 15); c. 4135G > A (exon 34);

c 1183A > T (exon 8)) (bang 2 vd 3) Id nhCrng dpt b i l n mdi phdt hi^n. Phdn bd cdc dOt bien IVS27 - 1G > A trong 5 gia dinh, p. F686S trong 4 gia dinh, eon lai mdi dOt b i l n (p R999X, e 1467 + 5G > A, p. R934X, p. S1387del, p. F6861, p. G1379S, p. I395F) xay ra trong mpt gia dinh. Den nay ed khoang trdn 150 ede dflt b i l n khde nhau cua gen ABCC8 vd tren 25 dOt b i l n khde nhau eua gen KCNJ11 da dupe bdo cao, phan bo dOt b i l n todn bo chieu ddi cua gen [9], Ddt b i l n ldn d gen ABCC8 vd KCNJ11 thudng gay ra b^nh canh ldm sdng ha glucose mdu cudng insulin ndng, [7].

Trong xdc dinh tuang quan k i l u gen vd mCrc dd ddp Crng dieu trj diazoxide, theo tdc gia K E Snider, 89% benh nhdn c6 dOt bien kdnh KATP d u a e md hoa bdi hai gen ABCC8 vd KCNJ11 la khdng e6 ddp Crng vdi d i l u tn bSng diazoxide vd ehi dat duae k i t qua kiem sodt glucose mdu tot khi d i l u tri phdu thu^t bdng cdt tyy, ngupe lai cdc benh nhdn mang cdc dot b i l n khde HNF4A, GLUD. HADH, UCP2 ed ddp Crng vdi diazoxide [8] Vdi nhdm benh nhdn chCing tdi 18/32 (56,2%) khdng phat hien ra ddt b i l n cdc gen thudng gdp.

TCNCYH 89 (4) - 2014

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Tjfligci^f NGHifeN C1>U ;Y H<afl|

trong nhdm ndy thi ddp Crng rat tot vdi dieu trj diazoxide. Mpt trudng hap 66t biln gen HNF4A ddp irng vdi dilu tri diazpxide vd mOt trudng dOt biln gen KCNJ11 ed ddp dng mdt phin vdi dilu trj va ed ha glucose mdu nhe sau dieu tri.

Trong nhdm ddt biln gen ASCC8, cdc trudng hpp ed hai ddt biln ldn vd mpt ddt bien trdi tCr b l thi 100% Id khdng ddp Crng vdi diazoxide vd can phai phSu thu$t cdt tyy 95%

vd chi mdt trudng hap trong s6 12 trudng hap phdt hi&n dup-c ddl bien gen ASCC8 cd mOt dot biln trdi ASCC8 tCr me vd khdng eIn phiu thu^t cdt tyy, ddp irng vdi dilu trj npi khoa.

V. K^T LUAN

Trdn nhung b$nh nhdn cudng insulin phdt hi$n dpt biln gen 43.8% trudng hp'p, trong dd ddt bien ABCC8 (37,6%), KCNJ11 (3,1%), HNF4A(3,1%}.

Cdc trudng hdP cd ddt biln gen ASCC8, d$c biet cd hai ddt biln ldn ho$e mpt ddt. bien trdi tCr b6 thi khdng ed ddp Crng vdi dilu tri ndi khoa va can phdu thu^t cdt tyy 95% sdm d l de phdng hg glucose mdu ndng.

Cdc trudng hap khdng ed dot biln gen thudng ddp li-ng vdi dieu tri ndi khoa.

L a i cam e n

Oe hodn thdnh de tdi ndy, tdi xin chdn thdnh cam antodn the cdc dong nghidp khoa Ndi t i l t - Chuyen hda Di truyen bdnh vl0n Nhi Trung uang dd sy giCip dd, gdp y vd tgo dieu kidn eho tdi tiln hdnh nghidn eCru. Tdi xm earn doan eac so lieu trong bdi bdo Id trung thyc vd chua duae ddng tr§n bat ky tap ehi ndo.

TAI LIEU THAM KHAO 1. Giurgea, I (2006). Molecular mecha- nisms of neonatal hyperinsulinism. Horm Res, 66(6), 289 - 296.

2. Pinney, S.E (2008) Clinical characteris- tics and biochemical mechanisms of congenital hyperinsulinism associated vi/ith dominant KATP channel mutations J Clin Invest, 118 (8), 2877 - 2886.

3. De Lonlay-Debeney, P (1999). Clinical features of 52 neonates v/ith hyperinsulinism".

NEnglJMed. 340(15), 1169-1175.

4. Hussain, K (2008). Diagnosis and man- agement of hyperlnsuliriaemie hypoglycaemia of infancy. Horm Res, 89(1), 2 -13

5. Ellard, S (2007). Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations Vifith opposite functional effects. Am J Hum Genet 81(2), 375-82.

6. Flanagan, S.E (2006). Mutations in KCNJ11, which encodes Kir6 2. are a com- mon cause of diabetes diagnosed in the first 6 months of life, vt'ith the phenotype determined by genotype. Diabetologia, 49(6), 1190 -1197.

7. James, C (2009). The genetic basis of congenital hyperinsulinism J Med Genet. 46 (5), 289 - 299.

8. Snider, K.E (2013). Genotype and phenotype correlations in 417 children with congenital hyperinsulinism. J Clin Endocrinol Metab, 98(2), E355 - 363.

9. Flanagan, S.E (2009). Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism. Hum Mutat, 30(2), 170 -180.

TCNCYH 89 (4) - 2014

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TAP CHI NGHIEN CU'U Y HQC Summary

MOLECULAR GENETICS, GENOTYPE AND PHENOTYPE CORRELATIONS OF CHILDREN WITH CONGENITAL HYPERINSULINISM

Objective of this study was to identify mutations in the ABCC8 and KCNJ11, HNF4A and GLUD genes, genotype and phenotype correlations of children with congenital hypennsulinism. A prospective study was conducted on 32 cases with congenital hyperinsulinism diagnosed and treated at the National Hospital of Pediatric from January 2010 to September 2012. The entena used for diagnosis was designed by Hussain K 2008. The results showed that 43.8% were detected with genes mutation, including ABCC8 (37,6%), KCNJ11 (3 1%), HNF4A (3 1%). 100%

cases who has two recessive mutations or one dominant gene mutation from the father of ABCC8, do not respond to diazoxide treatment and must undergo near total (95%) pancreatec- tomy, with other cases who do not have mutation usually respond to diazoxide. In conclusion, for children with congenital hyperinsulinism, it's necessary to take genetic testing to detect gene mutations in order to make an appropnate decision for medical treatment. Family with a child diagnosed with congepital hyperinsulinism need genetic counseling, prenatal diagnosis and follow up treatment immediately after delivery.

Key word: hyperinsulinism, hypoglycemia

TCNCYH 89 (4)-2014