Miclea D., Alkhzouz C., Popp R.A., Zimmermann A., Lazar C., Lazea C., Bucerzan S., Farcas M., Grigorescu-Sido P.

▪ Testarea genetica efectuata la Laboratorul de Genetica al Spitalului Clinic de Urgenţă pentru

Copii, Centrul Regional de Genetica Medicala Cluj

▪ Testarea genetica – diagnostic pozitiv intr-o patologie genetica

▪ Genetica medicala in urmatorii 15 ani – medicina genomica (medicina de precizie)

▪ Diagnostic si evolutie: boli rare, boli comune

▪ Tratament: Farmacogenetica

Ghiduri de testare genetica, in Europa - utilizate de ≈ 3-5 ani▪ Patologia endocrina: DSD/hipostatura – panel de gene NGS

▪ Boli genetice de metabolism: panel de gene NGS

▪ DD/ID, TSA, epilepsie – array CGH/SNP, panel, exom NGS

▪ Aceasta se poate efectua pentru tot mai multe patologii şi ȋn ţara noastră

1) patologia endocrină (Miclea D)▪ ADS/hiperplazia congenitala de corticosuprarenala şi anomalii ale pubertăţii

▪ hipostatura şi anumite displazii scheletale

▪ obezitatea

2) fibroza chistica şi anumite boli genetice de metabolism (Farcas M)▪ boala Gaucher, deficitul de alfa1antitripsină

3) patologia senzoriala (surditatea), psihiatrica şi neurologică (Farcas M, Miclea D)▪ RD/DI, TSA, epilepsia - forme izolate sau sindromice

▪ aparatura▪ Laboratorului de Genetica Medicala al Spitalului Clinic de Urgenta pentru Copii, Centrul Regional Cluj

▪ Laboratorului de Genetica Medicala, UMF Cluj

▪ Parteneriate cu:▪ Centrul Imogen, Cluj-Napoca

▪ Centrul de Medicina Genomica, Timisoara

▪ Colaborare cu:▪ Centrul Regional de Genetica Medicala, Dolj

▪ Spitalul Robert Debre (Dr AC Tabet) si Spitalul Bicetre (Dr J Bouligand), Paris, Franta

▪ Tehnici utilizate▪ Citogenetica clasica

▪ FISH

▪ PCR si variante

▪ Stripassay

▪ MLPA

▪ SNP array/CGH array

▪ NGS

▪Anomalii de dezvoltare sexuala

NEONATAL

▪ OGE ANORMALE

▪ Hipertrofie clitoridiana izolata

▪ Hipospadias posterior izolat

▪ Criptorhidie/ectopie testiculara unilaterala+micropenis

▪ Criptorhidie/ectopie testiculara unilaterala+hipospadias

▪ Criptorhidie/ectopie bilaterala: gonade palpabile in pozitie inghinala sau nici o gonada palpabila

Anomalii de dezvoltare sexuala

PUBERTAR

▪ OGE ANORMALE

▪ Virilizare OGE la momentul reactivarii axului gonadotrop

▪ Deficit 5 alfa reductaza, Deficit 17 cetoreductaza testiculara, ovotestis

▪ RETARD PUBERTAR

▪ Disgenezie gonadala (Turner, Klinefelter)

▪ Cariotip 45,X/46,XY

▪ AMENOREE PRIMARA

▪ Rezistenta completa la androgeni

▪ Dozari hormonale▪ 17OH progesteron

▪ DHEAS

▪ delta4 androstendion

▪ Testosteron

▪ AMH

▪ Ecografie gonade+OGI

▪ Cariotip+SRY

▪ Testare deficit 21OH

▪ Panel gene ADS

▪ Cariotip+SRY

▪ Deficit 21 hidroxilaza – stripassay 11 mutatii mai frecvente

▪ Panel gene:

▪ TruSight One 4800 gene OMIM morbide

Patient age(yrs)

social

gender phenotype Caryotype

Hormonal

investigation Ultrasounds SNP array NGS

p1 10male

Micropenis. 5th

clinodactyly 46,XY Testo N DHT ↓

N gonads, no

mullerian

derivates no pathogen CNV N

p2 18female

clitoridian

hypertrophy 46,XX N gonads, OGI VN no pathogen CNV N

p3 1female

Right inguinal

hernia. Feminin

OGE. 2 aunts

maternal line with

primary

amenhoree 46,XY N

left gonads in

inguinal conduct.

Absence muller

residus no pathogen CNV AR

p4 5male

Penoscrotal

hipospadias.

Craniofacial

dysmorphism.

Blefarophimosis.

Short stature. Aortic

bicuspidia. DSV.

Development delay 46,XY N

gonads N, no

mullerian residus VOUS CNV SYCE1 N

p5 7male

Micropenis.

Testicular

hypoplasia 46,XY N VOUS CNV SYCE1

p6 18male

Micropenis.

Pubertary delay 46,XY N CNV dup 16p11.2

p7 1female

clitoridian

hypertrophy 46,XX 17OHP↑ uterus Del CYP21A2

penoclitoridian

gland, labial

hypertrophy

fusionated

Patient age(yrs)

social

gender phenotype Caryotype

Hormonal

investigation Ultrasounds SNP array NGS

p9 3male

micropenis.

Hipospadias 46,XY N no muller residus no pathogen CNV

p10 20female

amenoree. Hirsutism.

Gonadal dysgenesis 46,XX N no pathogen CNV

p11 1female

clitoris hypertrophy,

labial hypertrophy,

vaginal and uretral

meatus presents,

right inguinal hernia 46,XY T DHT ↑

in inguinal hernia-

ovalary tumors with

testicule

appearance no pathogen CNV AR

p12 2male

proximal

hypospadias.

Criptorchidism 46,XY no pathogen CNV

p13 3female

gonadal dysgenesis,

craniofacial

dysmorphisms, short

4th and 5th

metacarpals, 5th

clinodactyly, SGA,

short stature 46,XY AMH, testo↓ no muller residus no pathogen CNV

p14 3male severe hypospadias 46,XY N no pathogen CNV MAMLD1

p15 14male

Penian hypospadias.

Right cryptorchidism.

Ginecomastia.

Pubertal

development. 46,XY N

no muller residus,

left testicule in

inguinal conduct, a

smaller tumor with

the same

localisation in right

side no pathogen CNV

p16 3male

Bilateral

cryptorchidism.

Gonadal dysgenesis 46,XY LH,FSH ↑↑↑

left scleroatrophic

testicule, ecografic,

absent right

testicule no pathogen CNV

penoscrotal

hypospadias. Scrotal

17 patients – 3 CNV VOUS/pathogenic

10 p15.3 p15.1 p14 p13 p12.31 p12.1 p11.22 p11.21 q11.21 q11.22 q11.23 q21.1 q21.2 q21.3 q22.1 q22.2 q22.3 q23.1 q23.31 q23.33 q24.2 q25.1 q25.2 q25.3 q26.13 q26.2 q26.3

Base Position 130.205.223 130.797.393 131.389.563 131.981.733 132.573.903 133.166.073 133.758.243 134.350.413 134.942.583

Cytogenetic Band q26.2 q26.3

Sequence (+) No sequence data file found for this chromosome.

CLRN3

PTPRE

PTPRE

MKI67

MKI67

MGMT

EBF3

GLRX3 TCERG1L PPP2R2D

PPP2R2D

PPP2R2D

BNIP3

JAKMIP3

DPYSL4

STK32C

LRRC27

LRRC27

LRRC27

LRRC27

LRRC27

PWWP2B

PWWP2B

C10orf91

INPP5A

NKX6-2

C10orf93

GPR123

KNDC1

UTF1

VENTX

MIR202

ADAM8

ADAM8

ADAM8

TUBGCP2

ZNF511

CALY

PRAP1

PRAP1

C10orf125

C10orf125

ECHS1

SPRN

LOC619207

CYP2E1

SYCE1

SYCE1

SYCE1

FRG2B

LOC728410

DUX4

LOC653548

LOC653544

LOC653543

LOC653545

LOC728410

DUX4

LOC653543

16 p13.3 p13.2 p13.13 p13.12 p13.11 p12.3 p12.2 p12.1 p11.2 p11.1 q11.1 q11.2 q12.1 q12.2 q21 q22.1 q22.2 q23.1 q23.2 q23.3 q24.1 q24.2q24.3

Base Position 28.672.615 29.071.335 29.470.055 29.868.775 30.267.495 30.666.215 31.064.935 31.463.655 31.862.375

Cytogenetic Band p11.2

Sequence (+) No sequence data file found for this chromosome.

SBK1

EIF3CL

EIF3C

EIF3C

CLN3

CLN3

APOB48R

IL27

NUPR1

NUPR1

CCDC101

SULT1A2

SULT1A2

SULT1A1

SULT1A1

SULT1A1

SULT1A1

SULT1A1

EIF3C

EIF3C

EIF3CL

ATXN2L

ATXN2L

ATXN2L

ATXN2L

ATXN2L

TUFM

SH2B1

SH2B1

SH2B1

SH2B1

SH2B1

ATP2A1

ATP2A1

RABEP2

CD19

SPNS1

LOC653390

RUNDC2C

LOC606724

BOLA2

BOLA2B

GIYD1

GIYD2

GIYD2

GIYD1

SULT1A3

SULT1A4

SULT1A3

SULT1A4

LOC388242

LOC388242

IMAA

MMAA

SPN

SPN

QPRT

C16orf54

ZG16

KIF22

MAZ

MAZ

PRRT2

C16orf53

MVP

MVP

CDIPT

LOC440356

LOC440356

SEZ6L2

SEZ6L2

SEZ6L2

ASPHD1

TMEM219

TMEM219

TAOK2

TAOK2

HIRIP3

INO80E

DOC2A

C16orf92

C16orf92

ALDOA

ALDOA

ALDOA

ALDOA

PPP4C

TBX6

CORO1A

LOC606724

BOLA2

BOLA2B

GIYD1

GIYD1

GIYD2

GIYD2

SULT1A4

LOC613037

LOC595101

IMAA

CD2BP2

CD2BP2

TBC1D10B

MYLPF

SEPT1

ZNF48

ZNF771

ZNF771

DCTPP1

SEPHS2

ITGAL

ITGAL

ZNF768

ZNF747

ZNF764

ZNF688

ZNF688

ZNF785

ZNF689

PRR14

FBRS

SRCAP

SNORA30

PHKG2

C16orf93

RNF40

ZNF629

BCL7C

MIR762

CTF1

CTF1

NCRNA00095

FBXL19

ORAI3

SETD1A

HSD3B7

HSD3B7

HSD3B7

STX1B

STX4

ZNF668

ZNF646

POL3S

VKORC1

VKORC1

BCKDK

BCKDK

MYST1

FUS

FUS

FUS

FUS

PYCARD

PYCARD

TRIM72

ITGAM

ITGAM

ITGAX

ITGAD

COX6A2

ZNF843

ARMC5

ARMC5

TGFB1I1

TGFB1I1

TGFB1I1

SLC5A2

C16orf58

ERAF

CSDAP1

C16orf67

ZNF720

ZNF267 HERC2P4

6 p24.3 p22.3 p22.1 p21.2 p21.1 p12.3 p12.1 q12 q13 q14.1 q14.3 q15 q16.1 q16.3 q21 q22.1 q22.31 q23.2 q23.3 q24.1 q24.3 q25.1q25.2 q25.3 q26 q27

Base Position 31.470.581 31.829.331 32.188.081 32.546.831 32.905.581 33.264.331 33.623.081 33.981.831 34.340.581 34.699.331

Cytogenetic Band p21.33 p21.32 p21.31

Sequence (+) No sequence data file found for this chromosome.

CCHCR1

CCHCR1

CCHCR1

TCF19

TCF19

POU5F1

POU5F1

PSORS1C3

HCG27

HLA-C

HLA-B

MICA

HCP5

HCG26

MICB

MCCD1

BAT1

BAT1

SNORD117

SNORD84

ATP6V1G2

ATP6V1G2

NFKBIL1

NFKBIL1

NFKBIL1

NFKBIL1

LTA

LTB

AIF1

BAT2

APOM

C6orf47

BAT4

CSNK2B

LY6G5C

LY6G6F

G6e

G6E

LY6G6D

LY6G6C

C6orf25

C6orf25

C6orf25

C6orf25

C6orf26

VARS

LSM2

HSPA1L

HSPA1A

HSPA1B

C6orf48

C6orf48

NEU1

SLC44A4

EHMT2

EHMT2

ZBTB12

C2

C2

CFB

RDBP

MIR1236

SKIV2L

DOM3Z

STK19

STK19

C4A

C4B

CYP21A2

CYP21A2

TNXB

TNXB

ATF6B

ATF6B

FKBPL

PRRT1

PPT2

PPT2

EGFL8

AGPAT1

AGPAT1

RNF5

RNF5

AGER

NOTCH4

C6orf10

BTNL2

HLA-DRA

HLA-DRB5

HLA DRB4

HLA-DRB1

HLA-DQA1

HLA-DQB1

HLA-DQA2

HLA-DQB2

HLA-DOB

TAP2

TAP2

PSMB8

PSMB8

TAP1

PSMB9

PSMB9

PPP1R2P1

HLA-DMB

HLA-DMA

BRD2

BRD2

HLA-DOA

HLA-DPA1

HLA-DPB1

HLA-DPB2

COL11A2

COL11A2

COL11A2

COL11A2

RXRB

SLC39A7

SLC39A7

HSD17B8

MIR219-1

RING1

VPS52

RPS18

B3GALT4

WDR46

TAPBP

ZBTB22

LYPLA2

KIFC1

PHF1

PHF1

PHF1

CUTA

CUTA

CUTA

CUTA

CUTA

SYNGAP1

ZBTB9

BAK1

GGNBP1

C6orf227

ITPR3

C6orf125

IP6K3

IP6K3

LEMD2

LEMD2

MLN

MLN

MIR1275

GRM4

HMGA1

HMGA1

HMGA1

HMGA1

HMGA1

HMGA1

C6orf1

C6orf1

C6orf1

NUDT3

RPS10

PACSIN1

SPDEF

C6orf106

C6orf106

Patient chr start stop size CNV Gene

Interpretatio

n

4 10 135257091 135378802 121711 DupSYCE1 VOUS

5 10 135252347 135378802 126455 DupSYCE1 VOUS

6 16 29595483 30192561 597078 Dup16p11.2 Pathogenic

7 6 32005904 32006896 992

Homo

delCYP21A2 Pathogenic

▪ 9 tested patients

▪ 6 patients

▪ MAMLD1 (Xq28): c.1066C>T exonic stopgain mutation

▪ Xlr

▪ Clinical picture – micropenis, hypospadias, bifid scrotum

▪ UMD predictor: pathogenic

▪ EXAC: 2 alleles for 121410 chromosomes

▪ AR gene - 2 patients

I. Forma centrala

Hipogonadism hipogonadotrop:

- tranzitor

- permanent

PANEL GENE

II. Forma periferica

Hipogonadism hipergonadotrop:

- afectare gonadala primara – intotdeauna patologic

- cel mai frecvent disgenezie gonadala secundara anomaliilor cromozomilor sexuali

CARIOTIP

▪ Suspiciune clinica daca• micropenis si/sau criptorhidism la nou-nascutii de sex masculin

• retardul pubertar la o varsta osoasa mai mare de 13 ani

▪ izolat (este afectat doar axul gonadotrop)

▪ 50% din cazuri cu anosmie/hipoosmie = sindrom Kallmann

▪ asociat altor afectari endocrine

▪ non-sindromic

▪ sindromic anomalii ale liniei mediane -despicaturi labio-palatine, agenezii dentare- anomalii ale urechii si surditate, agenezii renale, malformatii cardiace, anomalii scheletice ale extremitatilor, sinkinezii bimanuale

•HHC izolat – NGS – 9 pacienti• Panel gene: GNRHR, GNRH1, KISS1R, KISS1, TACR3, TAC3, KAL1, FGFR1, FGF8, PROKR2,

PROK2, WDR11, CHD7, SEMA3A, NSMF, HS6ST1, FSHB, LHB, SOX3, FGF17, IL17RD, DUSP6,SPRY4, FLRT3, PROP1, NR0B1, PCSK1, LHX4, HESX1, OTX2, RNF216, OTUD4, SOX2, POU1F1,SOX10, KALP, CUL4A, CUL4B, GNRH2, NRP1, NRP2, SIX6, PDYN, OPRK1, TAC1, TACR1,TACR2, NPVF, NPFFR1, PLXNA1, SEMA7A, LHX3, NPY, LHX2, POU2F1, POU3F2, SLIT2,ROBO3, LEPR, LEP, SLIT3, CGA, INHBA, PRLR, PCSK2, PLXNC1, DCC, ZIC1, LIFR, FARP2

•HHC+obezitate – MS-MLPA Prader Willi

▪ Cariotip

▪ Detectie premutatii FMR1

OBEZITATE

+▪ Hipotonie+tulburari de alimentatie la nou-nascut

▪ Hipostatura, anomalii scheletale (brahimetatarsie)

▪ Endocrinopatii: hipogonadism

▪ Retard psihomotor

▪ Tulburare vizuala, auditiva, retard limbaj

▪ Epilepsie, tulburare de comportament

▪ Sindrom dismorfic, sindrom malformativ

=>OBEZITATE GENETICA

▪ Prader Willi: MS-MLPA - deletii si UPD

▪ Obezitate+Retard mental: PCRq - CNV 16p11.2; 15q11.1

▪ Obezitate+retard mental+sindrom dismorfic+sindrom malformativ: MLPA microdeletii

▪ SNP array/CGH array – ideal!

HIPOSTATURA

Factori genetici > 80% Factori de mediu

• Factori etnici, talia parentala

Cresterea - Proces multifactorial

Factori de mediu!

Ultimii 150 ani – trend secular

HIPOSTATURA - CLASIFICARE• primara: afectiuni intrinseci ale osului

• Displazii scheletale (3%)

• Sindroame genetice (21%)

• RCIU fara recuperare (12%)

• secundara: afectiuni ce modifica fiziologia cartilajului de crestere

• Endocrine (11%)

• Afectiuni cronice (3%)

• Malnutritie, cauze metabolice, psihosociale

• Idiopatica (50%)

• Hipostatura Constitutionala (idiopatica), familiala

Lam WFF, et al., Chin Med J 2002:115, 607-6117

ACMG practice guideline: Genetic evaluation of short stature(Seaver LH and Irons M, 2009)

▪ La fete – cromozomi sexuali si SHOX - MLPA

▪ La baieti – 45,X/46,XY? – cariotip

Afectare endocrina – Hipostatura proportionata

▪ PROP1

▪ Ideal Panel gene

Afectare scheletala – Hipostatura disproportionata

▪ SHOX - MLPA

▪ FGFR3 - PCR

▪ Ideal panel de gene

▪ Daca exista suspiciune clinica

▪ test genetic tintit

▪ Daca nu exista suspiciune clinica

▪ Evaluare genomica: MLPA, SNP array, NGS – panel de gene, exom

SINDROM TURNER

SINDROM NOONAN

▪ PTPN11

from, Baraitser and Winter, Color Atlas of Congenital Malformation Syndromes, 1996

SINDROM RUSSELL-SILVER

▪ 10% - UPD 7mat

▪ Anomalii ale metilarii 11p15.5-35%

(from, Baraitser and Winter, Color Atlas of Congenital Malformation Syndromes, 1996)

SINDROM RUBINSTEIN-TAYBI

•

SINDROM WILLIAMS

• 99%: del 7q11.23

(from, Baraitser and Winter, Color Atlas of Congenital Malformation Syndromes, 1996)

PSEUDOHIPOPARATIROIDISM TIP IA

• GNAS1 anomalii ale metilarii

▪ FIBROZA CHISTICA

▪ BOLI GENETICE DE METABOLISM

Boala Gaucher detectia a 8 mutatii comune GBA prin metoda StripAssay

Deficit de alfa 1 antitripsina A1AT – identificarea alelelor M,S si Z

▪ SURDITATEA nonsindromica – 328 cazuri analizate

▪ GJB2 - 35delG, W24X

▪ GJB6 - D13S1830 si D13S1854

▪ MLPA

Testare c35delG

Homozigot heterozigot negativă

Testare pW24X

Homozigot Heterozigot negativă

(compus)

Testare GJB6

Homozigot Heterozigot negativă

(compus)

SECVENȚARE

Lazar C, Int J Pediatr Otorhinolaryngol, 2010

-X fragil

-cariotip (1200 cazuri analizate)

-MLPA microdeletii (430 cazuri analizate)

-SNP array/CGH array (210 cazuri analizate)

DIAGNOSTIC MAI BUN

▪ un randament diagnostic bun şi astfel un bun raport cost-beneficiu derivă din indicaţia adecvată a acestor teste –

centre de expertiza cu echipa multidisciplinara

▪ Cluj – Deficit 21 hidroxilaza, Boli lizozomale

▪ utilizarea ghidurilor pentru testare genetică!

▪ testarea genetica presupune din ce in ce mai mult folosirea unor tehnologii de analiza a genomului, utilizate

deja la noi in tara – pentru o interpretare buna – trebuie un numar limitat de patologii/centru

TRATAMENT MAI BUN

▪ intelegerea unui mecanism molecular va conduce la alegerea unei terapii de precizie (ex.tulburarile din

spectrul autist, oncologie)