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8.1 Diagnòstic Genètic Cromosòmic 2024 m36 A
8.1 Diagnòstic Genètic Cromosòmic 2024 m36 A
Aplicat a Medicina
Diagnòstic genètic
ATTG->ATAG ATTG->ATTAG
Karyotyping 5–10 Mb Cannot detect: small rearrangements below the resolution; nucleotide
variants; mosaicism < 10%a; UPDb
FISH ~100 kb Limited to the probes used (targeted analysis);
Cannot detect: nucleotide variants; mosaicism < 10%c; UPD
Array-based techniques ~20–200 kb Cannot detect: balanced rearrangement; mosaicism < 10%d; nucleotide
chromosomal microarray SNP-based variants; the nature of a structural aberration; independent cell lines;
array heterochromatic markers; triploidy (exception SNP array); UPD (exception
SNP array)
CNV detection in whole-exome 100 bp (exonic regions) – Cannot detect: balanced rearrangement; mosaicism < 18%f; the nature of the
sequencing ~150 kb (genome wide)e structural aberration; independent clones/cell lines
a
Hsu and Benn [7]; Hook [36]
b
Uniparental disomy
c
Wiktor and van Dyke [37]; Ballif et al. [38]; Mascarello et al. [39]
d
Vermeesch et al. [23]; Ballif et al. [38]
e
Pfundt et al. [32]
f
Pagnamenta et al. [40]
each technique must be taken into account (e.g., resolution, separation of pre- and post-PCR areas is required. Controls
reporting time) (Table 1). must be included in each PCR set-up to identify DNA or
PCR product contamination.
Sample preparation The most critical issues regarding sample preparation
Silva, M., de Leeuw, N., Mann, K. et al. European guidelines for constitutional cytogenomic analysis. Eur J Hum
involve ensuring, as much as possible, that the samples are
Genet 27, 1–16 (2019). https://doi.org/10.1038/s41431-018-0244-x
Depending on the referral reason, the tissue types and correctly identified (i.e., sample verification) and suitable for
sample preparation may vary. However, there are general the technique used, as well as minimising the possibility of
recommendations that should be considered when preparing misdiagnosis. For prenatal samples, a minimum of two cul-
samples for cytogenomic analysis. tures should be set-up and analysed, although for follow-up of
Cultures set-up for prenatal samples should be performed an abnormal rapid result only a single culture is required,
Mètodes diagnòstics en genètica
• Alteracions cromosòmiques
– Estudi
– Estudi mitjançant
mitjançant cariotip cariotip
– Permet
– Permet una visió
una visió general de la general
informació de la informació
genètica del genètica del
consultant
consultant
– Limitat
– Limitat a latècnica
a la capacitat capacitat
(>5Mb) tècnica (>5Mb)
– Detecta alteracions equilibrades
– Detecta alteracions equilibrades
• Alteracions gèniques
– Estudis
– Estudis moleculars
moleculars
Cal que
– saber
– Cal saber que (gen
busquem busquem
causal) (gen causal)
– Alta resolució (<5MB fins mutacions puntuals)
– Alta resolució (<5MB fins mutacions puntuals)
– Només investiga la zona interrogada
– Només investiga la zona interrogada
• Noves tecnologies à NGS
– Noves tecnologies permeten molts anàlisis i moltes
Noves tecnologies permeten molts anàlisis i moltes
dades dades
Tècniques diagnòstiques
Karyotyping 5–10 Mb Cannot detect: small rearrangements below the resolution; nucleotide
variants; mosaicism < 10%a; UPDb
FISH ~100 kb Limited to the probes used (targeted analysis);
Cannot detect: nucleotide variants; mosaicism < 10%c; UPD
Array-based techniques ~20–200 kb Cannot detect: balanced rearrangement; mosaicism < 10%d; nucleotide
chromosomal microarray SNP-based variants; the nature of a structural aberration; independent cell lines;
array heterochromatic markers; triploidy (exception SNP array); UPD (exception
SNP array)
CNV detection in whole-exome 100 bp (exonic regions) – Cannot detect: balanced rearrangement; mosaicism < 18%f; the nature of the
sequencing ~150 kb (genome wide)e structural aberration; independent clones/cell lines
a
Hsu and Benn [7]; Hook [36]
b
Uniparental disomy
c
Wiktor and van Dyke [37]; Ballif et al. [38]; Mascarello et al. [39]
d
Vermeesch et al. [23]; Ballif et al. [38]
e
Pfundt et al. [32]
f
Pagnamenta et al. [40]
each technique must be taken into account (e.g., resolution, separation of pre- and post-PCR areas is required. Controls
reporting time) (Table 1). must be included in each PCR set-up to identify DNA or
PCR product contamination.
Sample preparation The most critical issues regarding sample preparation
Silva, M., de Leeuw, N., Mann, K. et al. European guidelines for constitutional cytogenomic analysis. Eur J Hum
involve ensuring, as much as possible, that the samples are
Genet 27, 1–16 (2019). https://doi.org/10.1038/s41431-018-0244-x
Depending on the referral reason, the tissue types and correctly identified (i.e., sample verification) and suitable for
sample preparation may vary. However, there are general the technique used, as well as minimising the possibility of
recommendations that should be considered when preparing misdiagnosis. For prenatal samples, a minimum of two cul-
samples for cytogenomic analysis. tures should be set-up and analysed, although for follow-up of
Cultures set-up for prenatal samples should be performed an abnormal rapid result only a single culture is required,
Diagnòstic Cromosòmic
• Cariotip
– és la disposició ordenada dels cromosomes
– és la disposició ordenada dels cromosomes
del nucli del
d’unanucli
cèl·lulad’una
atenent lacèl·lula atenent la seva mida,
seva mida,
la posicióladelposició
centròmerdel centròmer
i el seu patró de i el seu patró de
bandes.
bandes.
– Ens permet veure tot el genoma..
– Ens permet veure tot el genoma..
– Limita la necessitat de cultiu cel·lularà
– Limita la necessitat de cultiu cel·lularà
retarda el diagnòstic
retarda el diagnòstic
– Tècnica manualà massa variabilitat
– Tècnica manualà massa variabilitat
A B
D E
G C G
Com obtenim un cariotip
• A partir de qualsevol teixit on les seves les
cèl·lules
• A partir siguin susceptibles
de qualsevol de divisió:
teixit on les seves les
– Sang
cèl·lules perifèrica
siguin susceptibles de divisió:
– Sang perifèrica
– Frotis
– Frotis mucosa mucosa
bucal obucal
pell o pell
– Medul·la
– Medul·la òssia òssia
– Líquid amniòtic
– Líquid amniòtic
– altres..
• Es – altres..
realitza un cultiu cel·lular a partir de les
• Es realitza
cèl·lules un cultiu
de la mostra cel·lular
i s’atura la divisióa partir de les
cèl·lules
cel·lular deenlal’etapa
(mitosi) mostra de ilas’atura
metafase. la divisió
cel·lular (mitosi) en l’etapa de la metafase.
Procediment per obtenir el cariotip
Afegir cèl·lules Inducció de mitosis
(sang, amnios, (PHA)
biòpsia) Transferir cèl·lules i
centrifugar per obtenir
Incubar 2-3 Aturar cèl·lules cèl·lules aïllades
dies en Metafase
(Colxicina)
Cultivar cèl·lules en medi ric a
37ºC i en atmosfera controlada
de CO2
Prepara cèl·lules en els
portaobjectes
Fixar les cèl·lules en
solució metanol-acètic
Karyotyping 5–10 Mb Cannot detect: small rearrangements below the resolution; nucleotide
variants; mosaicism < 10%a; UPDb
FISH ~100 kb Limited to the probes used (targeted analysis);
Cannot detect: nucleotide variants; mosaicism < 10%c; UPD
Array-based techniques ~20–200 kb Cannot detect: balanced rearrangement; mosaicism < 10%d; nucleotide
chromosomal microarray SNP-based variants; the nature of a structural aberration; independent cell lines;
array heterochromatic markers; triploidy (exception SNP array); UPD (exception
SNP array)
CNV detection in whole-exome 100 bp (exonic regions) – Cannot detect: balanced rearrangement; mosaicism < 18%f; the nature of the
sequencing ~150 kb (genome wide)e structural aberration; independent clones/cell lines
a
Hsu and Benn [7]; Hook [36]
b
Uniparental disomy
c
Wiktor and van Dyke [37]; Ballif et al. [38]; Mascarello et al. [39]
d
Vermeesch et al. [23]; Ballif et al. [38]
e
Pfundt et al. [32]
f
Pagnamenta et al. [40]
each technique must be taken into account (e.g., resolution, separation of pre- and post-PCR areas is required. Controls
reporting time) (Table 1). must be included in each PCR set-up to identify DNA or
PCR product contamination.
Sample preparation The most critical issues regarding sample preparation
Silva, M., de Leeuw, N., Mann, K. et al. European guidelines for constitutional cytogenomic analysis. Eur J Hum
involve ensuring, as much as possible, that the samples are
Genet 27, 1–16 (2019). https://doi.org/10.1038/s41431-018-0244-x
Depending on the referral reason, the tissue types and correctly identified (i.e., sample verification) and suitable for
sample preparation may vary. However, there are general the technique used, as well as minimising the possibility of
recommendations that should be considered when preparing misdiagnosis. For prenatal samples, a minimum of two cul-
samples for cytogenomic analysis. tures should be set-up and analysed, although for follow-up of
Cultures set-up for prenatal samples should be performed an abnormal rapid result only a single culture is required,
Citogenètica molecular
• • Aplicació de tècniques
Aplicació de tècniques de biologia de biologia molecular
molecular
aala la citogenètica
citogenètica
• • FISH (Hibridació
FISH (Hibridació in situ fluorescent)
in situ fluorescent) 80’s i 80’s i
90’s
90’s
– Sondes locus
– Sondes locus
– Centromèriques
– Centromèriques
– Telomèriques
– pintats
– Telomèriques
– pintats genòmica comparada) 2000’s
• CGH(Hibridació
• CGH(Hibridació genòmica comparada) 2000’s
– en portaobjectes
– en portaobjectes
– en arrays
FISH
FISH painting / Locus específic
Cr. X Cr. 13
LSI TP53
SO 17p13.1
SG D17Z1 SG
https://www.molecularcatalog.abbott/int/en/Vysis-TP53-CEP-17-FISH-Probe-Kit
FISH en Nucli interfasic
https://metasystems-probes.com/en/probes/xl/d-5060-100-og/
FISH en Nucli interfasic
LSI D12Z3 SG
D13S319 SO,
LAMP1 SA
Citogenètica molecular FISH
Avantatges: Problemes:
• • No calcultiu
No cal cultiu cel·lular
cel·lular à à • Només
• Només interroga
interroga les les
mésrapidesa
més rapidesa regions
regions conegudesconegudes
• • Podem veure
Podem veure l’alteració
l’alteració Cal saber
• saber
• Cal que es que
buscaes busca
sobre
sobre metafase
metafase (en el (en el (estudi
(estudi dirigit) dirigit)
cromosoma)
cromosoma) • Número
• Número limitat
limitat de colors de colors
• • Diagnòstics
Diagnòstics més més acurat
acurat de lesde les sondes
sondes (màxim 3)(màxim 3)
• • Nivell
Nivell dede resolució
resolució més més • Expertesa
• Expertesa moderada
moderada de de
elevat
elevat queque
convenciona
la della del cariotip
cariotip l’observador
l’observador
convencional
European guidelines for constitutional cytogenomic analysis 3
Karyotyping 5–10 Mb Cannot detect: small rearrangements below the resolution; nucleotide
variants; mosaicism < 10%a; UPDb
FISH ~100 kb Limited to the probes used (targeted analysis);
Cannot detect: nucleotide variants; mosaicism < 10%c; UPD
Array-based techniques ~20–200 kb Cannot detect: balanced rearrangement; mosaicism < 10%d; nucleotide
chromosomal microarray SNP-based variants; the nature of a structural aberration; independent cell lines;
array heterochromatic markers; triploidy (exception SNP array); UPD (exception
SNP array)
CNV detection in whole-exome 100 bp (exonic regions) – Cannot detect: balanced rearrangement; mosaicism < 18%f; the nature of the
sequencing ~150 kb (genome wide)e structural aberration; independent clones/cell lines
a
Hsu and Benn [7]; Hook [36]
b
Uniparental disomy
c
Wiktor and van Dyke [37]; Ballif et al. [38]; Mascarello et al. [39]
d
Vermeesch et al. [23]; Ballif et al. [38]
e
Pfundt et al. [32]
f
Pagnamenta et al. [40]
each technique must be taken into account (e.g., resolution, separation of pre- and post-PCR areas is required. Controls
reporting time) (Table 1). must be included in each PCR set-up to identify DNA or
PCR product contamination.
Sample preparation The most critical issues regarding sample preparation
Silva, M., de Leeuw, N., Mann, K. et al. European guidelines for constitutional cytogenomic analysis. Eur J Hum
involve ensuring, as much as possible, that the samples are
Genet 27, 1–16 (2019). https://doi.org/10.1038/s41431-018-0244-x
Depending on the referral reason, the tissue types and correctly identified (i.e., sample verification) and suitable for
sample preparation may vary. However, there are general the technique used, as well as minimising the possibility of
recommendations that should be considered when preparing misdiagnosis. For prenatal samples, a minimum of two cul-
samples for cytogenomic analysis. tures should be set-up and analysed, although for follow-up of
Cultures set-up for prenatal samples should be performed an abnormal rapid result only a single culture is required,
Hibridació Genòmica Comparada CGH
• Base teòrica de FISH
• Base teòrica de FISH
• Aplicat damunt de
metafase
• Aplicat damunt de de control
metafase de control
• Mira els guanys i les
• Mira els guanys i les
pèrduespèrdues de material
de material
cromosòmic
cromosòmic
• Expertesa
• Expertesa de de
l’observador i molt
l’observador i molt
entrenament
entrenament
Array-CGH (2000’s)
Sdr Wolf-Hirschorn
Del 4p16.
Dup 1q21.1
Sdr. Wolf-Hirschorn