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Disorders of the genome architecture: clinical diagnosis of constitutional disorders.

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Presentazione sul tema: "Disorders of the genome architecture: clinical diagnosis of constitutional disorders."— Transcript della presentazione:

1 Disorders of the genome architecture: clinical diagnosis of constitutional disorders

2 Genetic diseases are recognized to be one of the major categories of human disease Traditionally genetic diseases are subdivided: chromosomal (numerical or structural aberrations) monogenic or Mendelian diseases multifactorial/polygenic complex diseases epigenetics disorders mitochondrial genetic disorders

3 With the advent of newer molecular techniques, a number of new disorders and dysmorphic syndromes are delineated in detail Some of these conditions do not conform to the conventional inheritance patterns and mechanisms are often complex and unique

4 Examples submicroscopic microdeletions or microduplications trinucleotide repeat disorders epigenetic disorders due to genomic imprinting defective transcription or translation due to abnormal RNA patterning pathogenic association with single nucleotide polymorphisms copy number variations

5 Dhavendra Kumar, Genomic Med., 2009

6 Among these several apparently monogenic disorders result from non-allelic homologous recombination associated with the presence of low copy number repeats on either side of the critical locus or gene cluster.

7 Studi dei breakpoints molecolari sui riarrangiamenti cromosomici ricorrenti ha reso possibile capire le cause dei riarrangiamenti strutturali

8 Genomic rearrangements describe gross DNA changes of the size ranging from a couple of hundred base pairs, the size of an average exon, to megabases (Mb) When greater than 3 to 5 Mb, such changes are usually visible microscopically by chromosome studies

9 Genomic rearrangements describe mutational changes in the genome such as: duplication deletion insertion inversion translocation different from the traditional Watson-Crick base pair alterations

10 Genomic rearrangements can represent polymorphisms that are neutral in function, or they can also convey phenotypes via diverse mechanisms: changing the copy number (copy number variation or CNV) of dosage-sensitive genes disrupting genes creating fusion genes other mechanisms

11 The pathological conditions caused by genomic rearrangements are collectively defined as GENOMIC DISORDERS

12 Disordini genomici * dovuti a riarrangiamenti ricorrenti del DNA coinvolgenti regioni genomiche instabili ** il fenotipo clinico è la conseguenza dei dosaggio anomalo dei geni localizzati nella regione instabile

13 Selected disorders of genome architecture

14 Dhavendra Kumar, Genomic Med., 2009

15 Typically, the term 'genomic rearrangements' is only used to describe gross DNA changes ranging from thousands to sometimes millions of base pairs that can cover clusters of different genes Monogenic point mutations usually reflect errors of DNA replication and/or repair, whereas the gross genomic rearrangements are often caused by other mechanisms mediated or stimulated by genomic structural features genomic architecture

16 SINEs (short interspersed nuclear elements) LINEs (long interspersed nuclear elements) HERV (families of human endogenous retroviruses) The Alu repeat occurs about once every 3 kb in the human genome and includes examples that are transcribed The completion of human genome project has revealed that about 5% of the human genome is comprised of region- specific low-copy-repeats (LCRs)

17 Disease-causing genomic rearrangements can be recurrent, with a common size and fixed breakpoints (breakpoints cluster)

18 Recurrent genomic rearrangements Genomic diseases resulting from recurrent visible genomic rearrangements

19 Disease-causing genomic rearrangements can be non- recurrent with different sizes and distinct breakpoints for each event. The nonrecurrent rearrangements share a common genomic region-of-overlap, the smallest region of overlap (SRO), that encompasses the locus associated with the conveyed genomic disorder

20 Some of the non-recurrent rearrangements have one of their breakpoints localized in one small genomic region. This grouping of breakpoints is distinct from breakpoint clustering, but like clustering, it may reflect underlying genomic architecture (for example, palindrome or cruciform) important to the rearrangement mechanism

21 Three major mechanisms have been proposed for genomic rearrangements in the human genome: Non-allelic homologous recombination (NAHR) Non-homologous end-joining (NHEJ) Fork Stalling and Template Switching (FoSTeS)

22 Homologous, non allelic sequences, which include region-specific repeat gene clusters (LCRs), are intimately involved in causing chromosomal rearrangements

23 Identità di sequenza fra i dupliconi di 24 kb: >95% Lupski: 1991, 1992

24 Among the several genes located in the 1,4 Mb region between the two CMT1A-REP, only PMP22 is dosage sensitive

25 When the two LCRs are located on the same chromosome and in direct orientation, NAHR between them causes duplication and/or deletion.

26 When the two LCRs are located on the same chromosome but in opposite orientation, NAHR results in inversion of the fragment flanked by them

27 Interchromosomal and interchromatid NAHR between LCRs in direct orientation result in reciprocal duplication and deletion intrachromatid NAHR creates only deletion

28 Un numero crescente di malattie genetiche è causato da riarrangiamentiricorrenti del DNA che coinvolgono regioni instabili Questi riarrangiamenti portano a perdita o guadagno di geni sensibili al dosaggio Sono causati dalla presenza di dupliconi che fiancheggiano la regione di delezione o di duplicazione

29 Selected disorders of genome architecture

30 q11.23 Chromosome 7q deletion and Williams syndrome (circa 1:20.000) 7 (1,5 Mb) N.B.:Non identificabile con citogenetica classica

31 : Stenosi aortica sopravalvolare + t(6;7)(p21;q11.23) The elastin gene is disrupted by a translocation associated with suprvalvular aortic stenosis (Curran et al, Cell 73: , 1993)

32 The duplicons flanking the Williams-Beuren deletion at 7q Mb Identità di sequenza fra i dupliconi : >95%

33 Williams-Beuren: a contiguous gene syndrome Elastin haploinsufficiency: SVAS (supravalvular aortic stenosis) Mental retardation: ??? Peculiar behaviour: ???

34

35 La delezione 22q11.2 è associata alla sindrome di DiGeorge/ Velocardiofacial (1:4000 neonati)

36 - I disordini del sistema immunitario si manifestano soprattutto nellinfanzia e persistono fino alla giovane età portando a frequenti infezioni respiratorie - Con letà si rendono più evidenti i disturbi dello sviluppo, del comportamento e psichiatrici: ritardo mentale lieve, difficoltà di apprendimento, voce ipernasale, forte impulsività, ansia o fobie - Esiste una facies caratteristica di questa condizione (?): viso allungato, piramide nasale prominente con radice nasale rilevata e ali ipoplasiche, retrognatia, hooding delle palpebre superiori, rime palpebrali sottili e lievemente oblique in alto e in fuori

37 Genomic disorders: repeats complexity Chromosome 22q11-specific LCR (E. Beverly, 2000, 2001)

38

39 AA A A A AA AA A a La ricombinazione fra i geni e gli pseudogeni A rompe il gene del fattore VIII provocando emofilia di tipo grave

40 Il 99% dei casi di inversione del fattore VIII si originano alla meiosi del nonno materno

41 Genomic diseases resulting from recurrent visible genomic rearrangements

42

43 FISH with PWS/AS probes identifies the type of inv dup(15)

44 Normal phenotype Prader-Willi syndrome Angelman syndrome when associated with 15 UPD or 15q11-q13 deletion

45 48,XX,+mar,+mar: normal phenotype

46 Genomic diseases resulting from recurrent visible genomic rearrangements

47 inv dup(8p): more than 50 cases reported

48 * Hypotonia and development delay * Facial dysmorphism high forhead high forhead broad mouth with inferior prominent lip broad mouth with inferior prominent lip dysplastic big ears dysplastic big ears curly hair sparse in temporal area curly hair sparse in temporal area * Corpus callosum agenesis * Severe mental retardation Inv dup(8p)

49 The breakpoints of inv dup(8p) are remarkably constant Floridia et al, 1996: Am J Hum Genet and inverted

50 While building the contigs around the inv dup(8p) breakpoints, the first not deleted probe gave signals consistent with the olfactory receptors gene clusters localisation

51

52 Is there anything abnormal in the maternal apparently normal chromosomes 8?? FISH analysis was performed with probes internal to the REPs

53 In 4 of 12 (33%) families with a proband carrying the Williams -Beuren deletion, an heterozygous inversion at 7q11.23 was observed in the parent transmitting the disease-related chromosome Osborne et al, Nat Genet 29:321 (2001)

54 Osborne, NG, Nov. 2001

55 Alcuni riarrangiamenti cromosomici (quali linv dup 8p e la delezione 7q11.23) considerati de novo (i genitori risultano normali allanalisi citogenetica) si formano a causa di inversioni submicroscopiche presenti nel genitore alla cui meiosi si genera il cromosoma anomalo

56 What about translocations??

57 Regione Pseudoautosomica PAR1 Gene SRY, il primo determinante della differenziazione della gonade in testicolo X Y Alla meiosi I, i cromosomi X e Y ricombinano a livello di PAR1

58 Alla meiosi maschile, la traslocazione fra Xp e Yp porta alla formazione di 25% di spermatozoi con SRY traslocato allX e di 25% di spermatozoi con lY privo di SRY SRY traslocato su Xp

59 FISH con una sonda del gene SRY in un maschio 46,XX Xder(X)t(Xp22.3;Yp11.3) I maschi XX (circa 1:20.000) hanno un cariotipo femminile apparentemente normale. Lanalisi mediante FISH con una sonda specifica del gene SRY dimostra la trasposizione della regione distale di Yp su Xp. SRY

60 The polymorphic Yp submicroscopic inversion, already considered neutral, is the preferential background for the PRKX/PRKY translocation Mechanism of the Yp/Xp translocation at the basis of XX males and XY females X X Y Y

61 La traslocazione fra le porzioni distali di Yp e Xp avviene quasi sempre alla meiosi di maschi fenotipicamente normali portatori di uninversione submicroscopica di Yp distale. Tale inversione è un polimorfismo che dà suscettibilità al verificarsi della traslocazione

62 p16 p23 der(4) der(8)

63 The t(4;8)(p16;p23) translocation, either in the balanced or unbalanced form, has been reported in more than 14 de novo or familial cases. Taking into consideration that it may be undetected in routine cytogenetics, it could be the most frequent one after the t(11q;22q) which is itself the commonest translocation in man

64

65 Per definire a livello di sequenza, quali fossero i punti di rottura dei cromosomi 4 e 8, si è dovuto riempire i buchi fra i contigui presenti nelle relative mappe

66

67 Frequency of the chromosomes 4 heterozygous inversion in the population: 12% Frequency of the chromosomes 8 heterozygous inversion in the population: 26% Frequency of concurrent chromosomes 4 and 8 heterozygous inversion in the population: 2.5

68 All are mediated by duplicons and those benign represent genomic polymorphisms and give susceptibility to the formation of visible chromosome rearrangements In the last 10 years, seven recurrent submicroscopic inversions have been found: two pathological, five benign

69 An estimated 5-10% of the human genome consists of interspersed duplication that have arisen over the past 35 million years of evolution Many regions of the human genome likely contain LCR that predispose to rerrangements

70 Three major mechanisms have been proposed for genomic rearrangements in the human genome: Non-allelic homologous recombination (NAHR) Non-homologous end-joining (NHEJ) Fork Stalling and Template Switching (FoSTeS)

71 What about non-recurrent chromosome rearrangements? Non homologous end-joining (NHEJ) can be responsible for many of the non recurrent rearrangements.

72 In 21/33 SMS subjects with unusual deletion breakpoints the rearrangement occurs in LCR other than the SMS REPs Lupski, 2005 Evidence for NHEJ has been found by examining breakpoints for some deletions causing SMS This represents less than 20%–25% of SMS deletion cases

73 In presenza di: ritardo mentale/dismorfismi/anomalie congenite giustifica lanalisi FISH ………………………. …….previo accordo con il genetista/pediatra/neuropsichiatra visto il costo dellindagine ……consideriamo anche che oggi è possibile eseguire questanalisi mediante la tecnica MLPA ……..nuove tecniche??????

74 Lo studio delle anomalie cromosomiche molecular kariotyping Gli studi sui telomeri umani e il numero limitato di regioni genomiche note implicate nelle sindromi da microdelezioni dimostrano che microdelezioni e microduplicazioni non visibili con le analisi cromosomiche di routine sono la causa maggiore delle malformazioni umane e del ritardo mentale

75 Fino a pochissimo tempo fa era impossibile screenare lintero genoma umano per indivuare la presenza di submicroscopiche alterazioni di tipo numerico Recenti sviluppi nella tecnologia consentono lanalisi genoma per intero con una risoluzione senza precedenti, volte più grande di quella delle tecniche di routine Lo studio delle anomalie cromosomiche molecular kariotyping

76 1. Lo sviluppo di piattaforme microarray con un alto quantitativo di materiale di lavoro (cialde di vetro o silicone o perline di dimensioni microscopiche sulle quali centinaia di migliaia di sonde di acidi nucleici differenti vengono depositate o sintetizzate in un ordine predefinito) e la disponibilità di differenti format per analizzare specificatamente il DNA, lRNA o le proteine 2. Lottimizzazipone dei protocolli comparativi di ibridizzazione e dei sistemi di analisi dei dati Hanno permesso lo sviluppo della cosidetta tecnologia del karyotyping molecolare che consente la rilevazione sensibile e specifica di modificazioni di numero di regioni cromosomiali submicroscopiche lungo lintero genoma umano

77 Utilizzi del microarray Dalla sua introduzione nel 1997, la tecnica del microarray è stata utilizzata per 1. Studi nel campo dellevoluzione umana (2003) 2. Studio dei tempi di replicazione cellulare (2004) 3. Studio delle variazioni genomiche (2004) 4. Studio dei meccanismi patogentici, di sviluppo e progressione del cancro (2005)

78 Utilizzi del microarray Recentemente (2006……..) lo studio del DNA basato sulla tecnica del microarray è giunto nellambito della genetica clinica importante impatto nella diagnosi e nel counseling per i pazienti con ritardo mentale congenito e sindromi malformative, visto che le sue capacità di risoluzione nella ricerca delle anomalie cromosomiche vanno ben al di là di quelle delle tecniche di bandeggio tradizionali. Infatti…

79 Alterazioni numeriche submicroscopiche sia subtelomeriche che interstiziali sono responsabili di RM nel 5-20% dei casi Tali alterazioni submicroscopiche avvengono lungo tutto il genoma umano La frequenza di tali alterazioni congenite è inaspettatamente molto alta Quando testare i familiari? Quante alterazioni sono congenite e quante de novo?

80 Studi pilota: microarray e genetica clinica …….Ultimi due anni: solo un numero limitato di alterazioni può spiegare la maggior parte delle varietà nella popolazione umana (con un livello di risoluzione di 44 Kbasi) Queste regioni appaiono variare sia nei controlli che nei pazienti Tali sequenze potrebbero essere escluse dai protocolli diagnostici Si riduce considerevolmente il numero delle regioni che spiegano le variazioni di tipo numerico nel genoma umano

81 Conclusioni: molecular Vs traditional cytogentic technologies Molecular karyotyping Capacità di individuare alterazioni submicroscopiche causa di malattie DNA isolato direttamente dal sangue o da altro materiale biologico come tessuti bioptici Meccanismi interamente automatizzati, dal laboratorio fino allanalisi dei dati Possibilità di correlare in maniera molto specifica la alterazione genomica con la manifestazione fenotipica Traditional karyotyping Capacità di risoluzione limitata DNA isolato da cellule in replicazione Presenza di procedure manuali, sia in laboratorio che nellanalisi dei dati

82 Conclusioni: molecular Vs traditional cytogentic technologies Molecular karyotyping Non evidenzia riarrangiamenti bilanciati (inversioni e traslocazioni) Può non rilevare mosaicismi di basso livello Traditional karyotyping Capacità di evidenziare i riarrangiamenti bilanciati Rileva mosaicismi di basso livello Analisi cromosomiche di routine: sempre importanti nella diagnosi di RM e Malformazioni congenite

83 Array based comparative genome hybridization (array-CGH)

84 DIGESTIONE Usiamo gli enzimi ALUI e RSAI MARCATURA Per prima cosa aggiungiamo a ciascun campione 5μl di random primers (kit di marcatura).

85

86 Deletion/duplication at 7q11.23 last olig. Normal: 71,865 first olig. Del: 72,145 last olig. Del: 73,584 first olig. Normal:73,921 last olig. Normal: 71,865 first olig. Dup: 72,145 last olig. Dup: 73,584 first olig. Normal:73,921

87 a, b: coronal sections; c: axial section abnormal cortical development in cortical areas subserving language function

88

89 MR+ GH deficiency Three cases

90 Homozygous Deletion 17q23.3 of about 40 kb Mb Patients with IGHD type IA have complete absence of GH The majority of patients with type IA isolated GHD have large deletions within the GH-1 gene

91 A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism Bert B A de Vries, 2006 severe hypotonia from birth long hypotonic face ptosis blepharophimosis large, low-set ears tubular pear-shaped, nose with bulbous nasal tip, long columella with hypoplastic alae nasi broad chin long fingers nasal speech displayed an amiable and friendly disposition

92 appear The individuals had wide ventricles, as assessed by magnetic resonance imaging before the age of 1 year, and in succession appear additional periventricular white matter abnormalities. Evan E Eichler, Nat Genet, 2006

93 The 17q21.31 deletion encompasses two known genes, CRHR1 and MAPT and at least two putative genes, IMP5 and STH Gain-of-function mutations in MAPT, encoding the microtubule-associated protein TAU, cause autosomal dominant forms of frontotemporal dementia and parkinsonism

94 Haploinsufficiency for the microtubule-associated protein TAU may affect axonal elongation and neuronal migration, thereby explaining the major clinical features observed in the 17q21.31 microdeletion positive individuals Tau-deficient mice showed muscle weakness and memory disturbance

95 dup(X)(q28) (400 kb) last normal: 152, 661 Mb first duplicated: 152,692 Mb last duplicated: 153,130 Mb first normal: 153,148 Normal mother and mildly retarded sister with the same duplication In lymphocytes, skewed X inactivation in the females

96 When to apply array-CGH?? Only to selected cases?? To all mentally retarded subjects requiring cytogenetics analysis??

97 Laura: 22 years old, * Mildly delayed psychomotor development, * Mild broad base gait, * Verbal dyspraxia (poor speech clarity and simple grammatical construction that required a dedicated teacher at school) H 152cm. (10°-25° centile) W 54 (50° centile) CC 56 (90°-97° centile) When 19-years-old WAIS (Wechsler Adult intelligence Scale) test showed verbal IQ= 92; non verbal IQ= 86;

98 Genetic variation in the human genome Human Molecular Genetics, 2006, Vol. 15, Review

99 Large segments of the genome, ranging in size from 100 kb to ~3 Mb, varying several folds in copy number in the human population and denominated large-scale copy number variations (LCVs) A higher than expected association between LCVs and known segmental duplications has been noted (Iafrate et al. And Sebat et al. 2005) Copy-Number Variations

100 CNV influencing gene dosage, expression and disease Hum Mol Genetics, 2006, Vol. 15, Review

101 It is not clear to what extent such genomic changes are responsible for Mendelian or complex disease traits and common traits (including behavioral traits), or represent only benign polymorphic variation FCGR3 gene A recent study showed that copy number polymorphism in the FCGR3 gene predisposes to glomerulonephritis in humans and rats Aitman, T.J. Et al, Nature 2006, 439, 851–855

102 CCL3L1 gene CCL3L1 gene: the increased copy number has been shown to be protective against HIV infection Gonzalez et al, Science 2005 Specific categories of genes seem over-represented in CNVs including those important for interaction with the surrounding environment, such as olfaction and response to external stimuli Examples include: glutathione S-transferase genes glutathione S-transferase genes cytochrome P450 genes cytochrome P450 genes the complement component C4 the complement component C4 In each case, changes to gene copy number have been shown to give rise to concomitant changes in the level of enzyme activity, with phenotypic consequences.

103 Genomic Rearrangements and Phenotypic Traits Lupski and Stankiewicz, December 2005, Issue 6-e49

104 Genomic Rearrangements and Phenotypic Traits The two major mechanisms by which variation is introduced into our genome Such variations can be introduced by both endogenous and exogenous means These mutations can cause a disease trait if they affect gene structure, function, or regulation, as well as through the alteration of dosage Lupski and Stankiewicz, December 2005, Issue 6-e49

105 Il gratta e vinci del DNA prospettive attuali e future

106 genome-wide analysis had changed genomic medicine from the hypothesis-driven approaches to the relatively hypothesis-free genetic investigation no more which one is a good candidate gene for this syndrome? but have a look at the entire genome and see what is telling us about these individuals, in this situation

107 genome-wide analysis: 1) Array Comparative Hybridisation (aCGH) (or SNP Array)

108 The array interrogates in a single experiment thousands portions of DNA that cover the entire genome at very high resolution (100 kb, 20 kb, 1 kb, …) balanced rearrangements not detectable

109 same approach of conventional cytogenetics but resolution much higher with precise knowledge of gene content at unbalanced regions

110 genome-wide analysis: 2) Next Generation Sequencing or Massively Parallel Sequencing allows sequencing the entire genome or selected regions (exome sequencing) both balanced and unbalanced rearrangements detectable

111

112

113 Exome sequencing identifies the cause of a mendelian disorder S. Ng, NatureGenetics November 2009 four affected (Miller syndrome) individuals in three independent kindreds taking into consideration: nonsynonymous variants, splice acceptor and donor site mutations, and coding indels, with both a dominant and a recessive hypothesis filtering against dbSNPS and HapMap polyPhen (exclusion of mutations predicted to be benign)

114 X-chromosome exome sequencing (718 genes: 16 PAR, 702 X-specific) in 208 multiplex XLMR familes Tarpey et al, NG truncating variants recurrent in controls tr.var.: 10.6% of XLMR in less than 50% of the families the causative gene had been detected

115 when exome sequencing is negative, other classes of abnormalities have to be searched (regulatory mutations, copy number variants) what about the remaining 75% of XLMR families??? most but not all X-linked genes have been examined (technical problems)

116 exome sequencing (consumables: 1750 euros) unrelated affected individuals with largely overlapping phenotypes hypothesizing either AR-disease or AD de novo mutations AD- or X-linked diseases segregating in different members of a family (with complete or incomplete penetrance) AR-diseases when present in more than one member of the family in sporadic cases with genetically heterogeneous diseases for which several genes had already been identified (holoprosencephaly, ambiguous genitalia, epilepsy, retinitis pigmentosa, ……)

117 How exome sequencing might change the diagnostic approach (and medicine) specialists in different disciplines will be tempted to find the molecular bases of the diseases they are treating categories of patients will be collected not only clinically but also genetically homogeneous so allowing to better define onset and follow-up of that genomic alteration (disease) therapeutic drugs targeted to hit the pathway of altered proteins will be searched asymptomatic relatives will require to know their risk thus promoting research preventing or ameliorating the course of the disease

118 have a look at the entire genome and see what is telling us about these individuals, in this situation


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