Varianti Aplotipiche

1. RhD – nei caucasici è determinato da delezione di 1463 bp tra le Rhesus

Boxes del gene RHD , in altre popolazioni (Giapponesi e Africani neri) è
associato ad un gene RHD pressoché normale la motivazione della mancata
espressione è sconosciuta (nei Giapponesi)

2. RhD Ψ (66% RhD- Black Africans e 24% RhD- African Americans)

 10 esoni
 duplicazione di 37 bp che interessa gli ultimi 19nt dell’ introne 3 e i
primi 18 nt dell’ exon 4 (con possibile introduzione di un codone di
STOP)
 mutazione non senso (Y269X) nell’ exon 6
 4 SNP negli esoni 4 e 5.

3. RHD-CE-D

 Esoni 1 e 2, estremità 5’ dell’esone 3 ed esoni 9 e 10 di RHD

 estremità 3’ esone 3 ed esoni 4-8 di RHCE

NOTA BENE
Nella progettazione degli oligo bisogna ricordare che dato l’elevato grado di omologia
tra il gene RhD e RhCE c’è il rischio di ottenere falsi positivi (ho segnale positivo ma
è Rhd + o abbiamo amplificato RhCE??)
Diffrenze RHD/RHCE:
- Una sequenza presente in 3’UTR dell’esone 10 di RHD, non presente in
RHCE
- Una differenza di 600bp nell’esone 4 tra RhD e RhCE
- Differenze a carico dell’esone 7

REAL-TIME PCR MULTIPLEX:

 Esone 10 riconosce RdD(+), pseudo gene e gene ibrido

  Esoni 4 e 5 discriminano
a. RhD(+)/pseudo gene per la presenza di SNP in
quest’ultimo
b. RhD(+)/gene ibrido per l’assenza di tali esoni nella
variante.

ALLINEAMENTO OLIGO
ESONI 4-5 E 10 RHD

1…………………………………………………………………………………………………………………………………………………………………33300
33301 agcctgctgg gttgggctgg gtaagctctg aacaccagtc tcatggcttc aagtcacacc
33361 tcctaagtga agctctgaac tttctccaag gactatcagg gcttgccccg ggcagaggat
33421 gccgacactc actgctctta ctgggtttta ttgcagacag actaccacat gaacatgatg E4
33481 cacatctacg tgttcgcagc ctattttggg ctgtctgtgg cctggtgcct gccaaagcct
33541 ctacccgagg gaacggagga taaagatcag acagcaacga tacccagttt gtctgccatg
33601 ctgggtaagg acaaggtggg gtgagtggtc tcctacttgg gctgagcaga atggctcaga
33661 aaaggctctg gctgaaaaaa tctccctcct ttaccaagtt cccctgggtg tctgaagccc
33721 ttccatcatg attcatttct ttgagtagtg tttgctaaat tcataccttt gaattaagca
33781 cttcacagag caggttcagg aggcctgggg tatgcagatt tcaaccctct tggcctttgt
33841 ttccttgtct gtaaaatgtg gttagctggt atcagcttga gagctcggag gggagacgtg
33901 acttccccat ctaactctaa gtgacaaggc tgagactctc cagccctagg attctcatcc
33961 aaaacccctc gaggctcaga cctttggagc aggagtgtga ttctggccaa ccaccctctc
34021 tggcccccag gcgccctctt cttgtggatg ttctggccaa gtttcaactc tgctctgctg E5
34081 agaagtccaa tcgaaaggaa gaatgccgtg ttcaacacct actatgctgt agcagtcagc
34141 gtggtgacag ccatctcagg gtcatccttg gctcaccccc aagggaagat cagcaaggtg
34201 agcagggcgc tgcccttggg cagcacttgg gtctaacagg actagcacac atatttatgc
34261 ccctccccac cccagggcca gcgtgggttg ggagagggca tgccgggtgg tggagctgtg
34260…………………………………………………………………………………………………………………………………………………………………61260
61261 cctatatatc caagatctct tccaattcag attttatgaa agaatttcta aggtctttgt
61321 aatgagacat ttaggctgtt tcaagagatc aagccaaaat cagtatgtgg gttcatctgc
61381 aataaaaatg tttgttttgc ttttacagtt tcctcatttg gctgttggat tttaagcaaa
61441 agcatccaag aaaaacaagg cctgttcaaa aacaagacaa cttcctctca ctgttgcctg
61501 catttgtacg tgagaaacgc tcatgacagc aaagtctcca atgttcgcgc aggcactgga
61561 gtcagagaaa atggagttga atcctttctc tgccactctt tgaggagaat ctcaccattt E10
61621 attatgcact gtagaataca acaataaaat acagccatgt accacataac aacatcttgg
61681 taaacaacag actgcatata tgatggtggt catccagtaa gctaaggtta atttattatt
61741 attccttgtt tttttttttt tttttttttt tttgagatgt agtcttactc tgtcacccag
61801 gctagagtgc aatggcacca tcttggctca ctgcaacctc tacctcctgg gttcaagcaa
61861 atctcctgcc tcagcctcca aagtagctgg gattacaggc acccaccaca tctggctaat
61921 tttttgtatt tttagtaaag atggggtttc accatgttgg ccaggctgat ctcaaactcc
61981 tgacctcaag tgatctgccc gcctcggcct cccaaagtgc tggaaccaca ggcctgagcc
62041 actgtgccca gccttgtttg cttttttaac agataacagt gtgctcatag aaactgcttt
62101 gacatgactg caatcatgtg cttcatagaa acttaattag attataccac tagagtcttc
62161 agatttttat actttttttt tttgaaacgg agtctcactc tgtcaccagg ctggagtgca
62221 gtgccgcaat ctcggctcac tgcaacctcc gcctcccagg ttcaagcaat tctcctgcct
62281 cagcctcccg agtagctgga attacaagtg cgcactacca cacccagcta atttttgcat
62341 ttttacttga cagggtttca ccatgttggc taggatagtt tcaccaggat ctcttggcct
62401 catgatcagc ctgcctcggc ctcccaaagt gctgggatta caggtgtgag ccaccgtgcc
62461 cagcctatac ttcccttttt gaataccatt tggtgttttg aagaattaac agctttgtga
62521 acgtggcagt gcttgtgatt caggcttcca ttgagaccaa ggggagaacc tggttgcagg
62581 acaaacagac ggacagcgtg tggcagtgtt taaatgctct tctgaaggct gatacgacag
62641 ctctctgtgc actgattgca tatgcatccc aagattatat tattgttttc tactgctatg
62701 tgtcacactt tgccaaacag gatgtggaaa atgaataagc ggttttctta ggcacttctt
 62761 aacagacaat tggtcaaaat gaactccatt gcttaagaaa cacataaaca ccatttagtc
62821 actgaacata gctatatgta tggttgttac tatgggaaat cttgttttgc caattttctt
62881 tgaaaattct ggcagaccaa ggttcttttt gtttacataa tacttgaaaa ataaaaatga
62941 acaagctaac aaactaccaa gttttcactt acataaatgt agttgcatac agaaaatgtg
63001 actgtgaatt aatttttcta ggacttttaa actataagca ctatttgcac aaaagagaac
63060…………………………………………………………………………………………………………………………………………………………………64956

FONTI
“PREDICTION OF FETAL D STATUS FROM MATERNAL PLASMA: INTRODUCTION OF A
NEW NON-INVASIVE FETAL RHD GENOTYPING SERVICE”
K.M. Finning, P.G. Martin, P.W. Soothill, and N.D. Avent

RHD exon 4 EX4F CTGCCAAAGCCTCTACAGG

EX4R ATGGCAGACAAACTGGGTGTC*
EX4P TTGCTGTCTGATCTTTATCCTCCGTTCCCT

RHD exon 5 EX5F CGCCCTCTTCTTGTGGATG*

EX5R GAACACGGCATTCTTCCTTTC
EX5P TCTGGCCAAGTT*TCAACTC*TGCTCGCT

Le basi sottolineate sono mismatch, usati per aumentare la specificità dei primers.
Le basi in grassetto distinguono RhD da RHCE
Le basi con asterisco distinguono RhD da RhD Ψ.

“PRENATAL DIAGNOSIS OF FETAL RHD STATUS BY MOLECULAR ANALYSIS OF

MATERNAL PLASMA”

RHD exon 10 EX10F CCTCTCACTGTTGCCTGCATT

EX10R AGTGCCTGCGCGAACATT
EX10P (FAM) TACGTGAGAAACGCTCATGACAGCAAAGTCT
(TAMRA)