Miclea D, Zimmermann A, Popp RA, Lazea C, Al-Khzouz C ...ƒți de... · STUDII DE EVALUARE A...
Transcript of Miclea D, Zimmermann A, Popp RA, Lazea C, Al-Khzouz C ...ƒți de... · STUDII DE EVALUARE A...
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Miclea D, Zimmermann A, Popp RA, Lazea C, Al-Khzouz C, Bucerzan S, Grigorescu-Sido P.
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Centrul Regional de Genetica Medicala, Cluj
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Deficit 21 hidroxilaza – Secventiere/Stripassay 63 cazuri confirmate
Alte anomalii de dezvoltare sexuala:
Teste genetice
Cariotip+SRY – 87 cazuri
NGS: panel de gene – 10 cazuri
Analiza prin microarray - 12 cazuri
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NGS: 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
10 cazuri analizate – 5 variante patogene
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Cariotip
Premutatii FMR1 – mamele pacientilor cu sindrom X fragil
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Displazii scheletale:
SHOX: FISH/MLPA – 87 cazuri analizate
FGFR3: PCR-RFLP – 115 cazuri analizate
Alte cauze de hipostatura:
PROP1: PCR-RFLP – 92 cazuri analizate
Russell Silver: MS-MLPA – 10 cazuri analizate
Sindrom Noonan(PTPN11): PCR-RFLP – 39 cazuri analizate
Microdeletii care asociaza hipostatura: MLPA – 50 cazuri analizate
Pseudohipoparatiroidism 1a (osteodistrofie ereditara Albright) GNAS
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Prader-Willi: metilare/deletii – FISH/MLPA – 63 cazuri
CNV: 16p11.2, 15q11.2 – PCR cantitativ
Sindrom Sotos: NSD1 – FISH/MLPA – 10 cazuri
Pseudohipoparatiroidism tip 1A – amprentare gena GNAS
Obezitatea si retard mental: analiza prin microarray – 36 cazuri
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AnaMaria, 3 ani 4 luni
MI: nefrocalcinoza (ecografie abdominala – dureri abdominale)
APF: VG 38 SA, G=3200g; L=50 cm, dezvoltare somatica si neuropsihomotorie corespunzatoare
APP: fara semnificatie
AHC: mama si bunic patern – litiaza renala
Vigantol 1000 ui/zi 1-18 luni si sezonul rece
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T=98 cm (+1ds)
G=12 kg (-3ds pt talie)
Fara deformari osoase
Deshidratare grad 1: buze uscate
Aport lichidian = 2000 ml/zi
Diureza = 1500 ml/zi
TA=90/60 mmHg
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Ecografia renala: nefrocalcinoza medulara
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Sange
Ca total = 10,5 mg/dl(8,8-10,4) ↑ Ca corectat = 10.42 mg/dl
Ca2+ = 4.81 mg/dl (4.2-5.2)
PO4 = 1.45 mmol/l(1.1-1.94)
PTH – 15,4 pg/ml (vn 15-65) N/↓
25 OH Vit D3 = 31.5 ng/ml (vn 30-100 ng/ml)
1.25 (OH)2 vit.D3 = 103 (vn 22-111) N/↑
Fosfataza alcalina =268 UI/L (vn≤281 UI/L)
Ionograma: Na, K, Mg- VN
Uree = 40 mg/dl (16.6-48.5)
Creatinina = 0.42 mg/dl (<0.47)
Cromatografia Aa urinari VN
TSH = 1.96 mUI/l (0.7-5.97)
T4 liber = 17.8 pmol/l (12.3-22.8)
Astrup VN
Urina
Ca/creatinina u = 0.9 (vn<0.5) ↑
PO4 = 24 mg/kg/24h (vn 15-30)
Ionograma urinara: Na, K, Mg - VN
Ph = 7
Densitate = 1016
Examen de urina – cristale de fosfat amorf
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Hiperparatiroidism – NU (PTH adaptat)
Intoxicatie cu vitamin D – NU 25OH VN
Sinteza neadaptata a 1,25 OH DA
Mutatie CYP24A1 – degradare 1,25 OH
Hipersensibilitatea la vitamina D
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Densitometrie osoasa:
L1-L4 -0.2
Col femural -0.1
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Nefrocalcinoza (fara afectare osoasa)
Hipercalcemie si hipercalciurie
Hipersensibilitate la vitamina D? (hipercalcemie cu PTH scazut/adaptat)
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NGS (secventiere multipla paralela) – panel de gene – metabolism fosfocalcic
AP2S1, CASR, CLCN5, CYP24A1, CYP27B1, CYP2R1, CYP3A4, DMP1, ENPP1, FAM111A, FAM20A, FGF23, GALNT3, GC, GNA11, GNAS, GTF2I, HDAC4, IHH, KL, PDE4D, PDIA3, PHEX, PRKAR1A, PTH, PTH1R, PTHLH, SAMD9, SLC34A1, SLC34A3, SLC9A3R1, TBCE, TRPC3, TRPS1, VDR
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Pacienta – heterozigot compus SLC34A3_ex4 – c.304+2T>C – heterozigot
SLC34A3_ex13 – c.1462G>C – heterozigot
Mama- heterozigot SLC34A3_ex4 – c.304+2T>C – heterozigot
Varianta probabil patogena, rara – afecteaza probabil matisarea ADN (descrisa de Lorenz Depiereux et al 2006)
Tata - heterozigot SLC34A3_ex13 – c.1462G>C – heterozigot
Varianta nedescrisa pana acum, prezisa patogena de algoritmurile de predictie informatica
STUDII DE EVALUARE A METABOLITILOR VITAMINEI D (Spectrometrie de masa) in curs
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proteina transportoare a fosfatului dependenta de Na tip2 NaPi-IIc
AR
Fiziopatologie: Pacientii prezinta hipofosfatemie prin pierdere renala de fosfati
1,25(OH)2 ↑ - prin stimulare 1α la nivel renal (prin hipofosfatemie)
Tablou clinic CLASIC: rahitism hipofosfatemic cu hipercalciurie
asociaza in general o simptomatologie osoasa – rahitism Dar, formele cu expresie renala pura sunt posibile
Uneori, fenotip de hipersensibilitate la vitamina D
Daca mutatii heterozigote fenotip nu f cunoscut
posibil risc crescut de litiaza renala (factor de risc) Dasgupta et al 2014 J Am Soc Nephrol
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inhiba 1α hidroxilaza/fosfati
Se administreaza intrucat nefrocalcinoza (f severa de hipersensibilitate vit D)
Tratament Ketoconazol 3 mg/kg/8h
Inhiba 1α hidroxilaza
Daca hipersensibilitate la vitamina D benigna Doar monitorizare
hiperhidratare/dieta adaptata in calciu
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AVANTAJE SI LIMITE ALE TEHNICILOR GENETICE DE NOUA GENERATIE
Abordarea diagnostica
“Phenotype first?” sau “Genotype first?” sau
AMBELE
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• Tendinta actuala spre o abordare genomica a patologiilor genetice
• Microarray: CGH array, SNP array
• Secventiere masiva paralela (NGS): tintita (panel de gene), exom,
genom
• Utilizate pe scara larga in lume in special dupa anul 2010, initial in
cercetare dar acum si pt diagostic – ghiduri de diagnostic (array 2010,
NGS in curs)
• Deschid calea spre o abordare noua: “genotype first”
1. testarea genetica si stabilirea genotipului
2. explicarea fenotipului pornind de la genotip
*Miller et al 2010
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• in patologii complexe, poligenice
• Pleiotropia, expresivitatea variabila, penetranta incompleta
• Nu e necesar dg clinic specific
• Permite un diagnostic presimptomatic
• Identifica noi sindroame
DAR
• Poate dezvalui informatii nedorite la varsta pediatrica
• deseori variante cu semnificatie incerta
• Uneori, scaderea interesului pentru o abordare clinica mai amanuntita
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• Un bun clinician poate intui intr-un anumit numar de cazuri un diagnostic genetic
• in special in bolile monogenice cu penetranta completa, fara variabilitate clinica marcata
• Aceasta abordare scade costurile testarii genetice, daca evaluarea este tintita
DAR, uneori
• expresivitatea variabila
• pleiotropia
• penetranta incompleta
• bolile complexe multifactoriale
• oligogenismul
nu permit orientarea dg etiologic
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• Un dg clinic bun + un diagnostic genetic bun
• O analiza tintita phenotype first cand este posibil
• Cand nu - abordare genotype first
• Rezultatele genetice nu se pot interpreta in absenta fenotipului
• PRESUPUNE INTALNIRI PERIODICE CLINICIAN – GENETICIAN - DISCUTII ASUPRA FO PACIENTI
• prin tehnici de noua generatie (devin tot mai putin costisitoare, dar implica resursa umana pregatita) • array: 200 euro
• exom: 500 euro
• genom: 800 euro
• O abordare in tandem fenotip - genotip scade costurile
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SECVENTIEREA MULTIPLA PARALELA - NGS • Se bazeaza pe lectura a milioane de secvente ADN in paralel
• Cost din ce in ce mai mic
• Genomica: panel de gene, exom, genom
• Transcriptomica, epigenomica
• Multe variante per exom, circa 60.000 – adnotarea lor: benigne, VUS, patogene
• Indicatii NGS: in general in boli mendeliene
• TSA, deficit intelectual (dg 50% din cazuri)
• Endocrinologie (hgonadismhgonadotrop 50% cazuri)
• Malformatii cardiace congenitale (30% din cazuri)
• Boli genetice de metabolism
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INTERPRETAREA DATELOR - BIOINFORMATICA
Insa testele genomice necesita o platforma bioinformatica bine pusa la punct
Multe variante, CNV-uri
Adnotarea lor cu ajutorul bazelor de date existente prin intermediul platformelor bioinformatice (Galaxy – NGS, Cartagenia - array)
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testarea genetica in patologia endocrina: anomalii de dezvoltare sexuala, hipogonadism
hipogonadotrop, insuficienta ovariana prematura, hipostatura, obezitatea sindromica
ABORDARE PHENOTYPE AND GENOTYPE FIRST!
Presupune un parteneriat clinician – genetician pentru interpretarea variantelor identificate prin testare genetica
Adnotarea variantelor genetice prespune o munca de cercetare, incununata de stabilirea unui diagnostic
in viitor, intelegerea unui mecanism molecular va conduce la alegerea unei terapii personalizate
presupune folosirea unor tehnologii de analiza a genomului, disponibile la noi in tara
UNEORI DISTINCTIA BOALA RARA - BOALA COMUNA, ETIOLOGIC VORBIND, ESTE IMPOSIBILA