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«Improvement of MLPA technique (Multiplex Ligation-dependent Probe Amplification) to study human genetic diseases Doctoral Dissertation Eva ...»

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MASARYK UNIVERSITY

Faculty of Science

Institute of Experimental Biology

Improvement of MLPA technique

(Multiplex Ligation-dependent Probe Amplification)

to study human genetic diseases

Doctoral Dissertation

Eva Hladílková

Supervisor: Associate Professor Petr Kuglík Brno 2012

BIBLIOGRAPHIC ENTRY

Author: MSc. Eva Hladílková née Zrnová

Faculty of Science, Masaryk University Institute of Experimental Biology Department of Genetics and Molecular Biology Title of the dissertation: Improvement of MLPA technique (Multiplex Ligationdependent Probe Amplification) to study human genetic diseases Degree programme: Biology Field of study: General and Molecular Genetics Supervisor: Associate Professor Petr Kuglík Year of defense: 2012 Language used: English Key words: Chromosomal aberrations, mental retardation, molecular cytogenetics, MLPA, array-CGH

BIBLIOGRAFICKÝ ZÁZNAM

Autor: Mgr. Eva Hladílková roz. Zrnová Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Oddělení genetiky a molekulární biologie Název disertační práce: Využití techniky MLPA (Multiplex Ligation-dependent Probe Amplification) při studiu geneticky podmíněných onemocnění člověka Studijní program: Biologie Studijní obor: Obecná a molekulární genetika Školitel: doc. RNDr. Petr Kuglík, CSc.

Rok obhajoby: 2012 Jazyk: Angličtina Klíčová slova: Chromozomové aberace, mentální retardace, molekulární cytogenetika, MLPA, array-CGH © Eva Hladílková, Masarykova univerzita, 2012

ACKNOWLEDGEMENT

I would like to thank to everybody, who intentionally or unintentionally contributed to my dissertation thesis. First of all, I thank my supervisor associate professor Petr Kuglík for expert advice, comments and support during my whole study. I would like to thank all colleagues from Integrated Laboratory of Molecular Cytogenetics at Department of Medical Genetics, University Hospital Brno and colleagues from Department of Medical Genetics, Ullevål University Hospital, Oslo for creating a very pleasant working environment. Last but not least, I thank my family and my husband for their support during my whole study.

The experiments were predominantly done at Integrated Laboratory of Molecular Cytogenetics at Department of Medical Genetics, University Hospital Brno, the Czech Republic in cooperation with Faculty of Science, Masaryk University Brno and Czech Myeloma Group at Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno and with Department of Medical Genetics, Ullevål University Hospital, Oslo, Norway.

The experimental part of the dissertation thesis was supported by MSM0021622415 from the Ministry of Education, Youth and Sports, the Czech Republic; by Research Council of Norway, The Yggdrasil mobility programme (202752); by European Science Foundation (ESF) 'Frontiers of Functional Genomics' (3419).

ABSTRACT

Unbalanced structural chromosomal aberrations (duplications and deletions) play a major role in the pathogenesis of many genomic diseases in human. Due to this fact, new high sensitive methods revealing gains and losses of the genetic material hold an important position in modern molecular-cytogenetics. Mental retardation (MR) as a permanent and incurable disease affects about 2% of newly born children. Because the cause of the MR is still unknown in an approximately half of the cases, MR has become a frequent focus of genetic research. Due to the development of sensitive molecular cytogenetic methods, it was proven that chromosomal aberrations in subtelomeric regions are responsible for idiopathic MR in up to 5% of the cases. Since 2002, an approach based on the classic PCR reaction in a conjunction with amplification of ligated probes (MLPA - Multiplex Ligationdependent Probe Amplification), has been developed for analysis of DNA sequences.

As part of my dissertation, MLPA has been successfully implemented and optimized for subtelomeric screening into the clinical laboratory practice at the Integrated Laboratory of Molecular Cytogenetics, University Hospital Brno, in patients with unexplained MR and developmental delay (DD). The specific correlation genotype/phenotype was determined in 14 patients with detected chromosomal imbalances. It was proposed an algorithm for a diagnostic process in patient with unexplained MR/DD, dysmorphic features and congenital defects which is based on combination of all available diagnostic tools at Department of Medical Genetics according their cost-effectivity and reliability in our conditions.

In this study, MLPA assay was used to search for pathogenic subtelomeric CNVs in a cohort of 233 patients with unexplained MR/DD. These patients have been investigated at the Department of Medical Genetics between years 2007 - 2010. My research was conducted by using the MLPA method with kits number P036 and P070, which are designed for subtelomeric screening. The chromosomal rearrangement was detected at 14 out of 233 cases. The detected CNVs can be divided into the following groups: clearly pathogenic CNVs that overlap known microdeletion syndromes (Microdeletion syndrome 1p36 - Cases 1, 4, 6 and the Microdeletion syndrome 22q13 - Cases 2, 3, 9, 13), CNVs that overlap pathological chromosomal region identified in other studies (in Cases 8, 11), potentially pathogenic CNVs (in Cases 5, 7, 12, 14) and polymorphic CNVs (in one incident - Case 10).





Record of these changes (14/233) was 6%, which corresponds with the worldwide described detection rate of subtelomeric chromosomal aberrations in patients with unexplained MR/DD. This confirmed that MLPA revealed additional 6% of chromosomal aberrations, which could be cause of MR/DD. The use of MLPA it the clinical laboratory practice increased the detection rate of chromosomal aberrations and so the clarification of the causes of idiopathic MR/DD.

However new genome-wide copy-number detection using array-CGH is becoming more effective for the investigation of patients with MR/DD, the implementation of this method into routine clinical laboratory practice still presents funding problems and challenge with the difficult clinical interpretation of the results. My study has shown MLPA to be an alternative diagnostic tool for the detection of cryptic CNVs for cytogenetic laboratories that have not (yet) access to more sophisticated array-based technology.

In conclusion, my results show that the implementation of new techniques such as MLPA, coupled with array-CGH or FISH analysis to the routine diagnostic screening algorithm in patients with unexplained MR/DD, would definitely help in identifying of the subtelomeric chromosomal aberrations, which otherwise could be missed in the conventional G-banded karyotyping. MLPA takes main place in routine screening of subtelomeric changes as well as screening of the most frequent microdeletions in patients with negative G-banding findings. Specialized high-resolution techniques are certainly more powerful tools in establishing the proper phenotype/genotype correlations and the determination of candidate genes. This helps to provide accurate genetic counselling to the affected families, also for an improved prevention and possible therapies in patients (physiotherapy, psychotherapy, and pharmacotherapy). Eventually, more families will be informed about the cause of the disease of their family member that allows targeted prenatal or preimplantation diagnostics. MLPA has become reliable and cost-effective diagnostic tool at the Department of Medical Genetics, University Hospital Brno. The subtelomeric screening by MLPA is now performed up to 60 patients with unexplained MR/DD or additional congenital abnormalities a year.

ABSTRAKT

Strukturní chromozomové aberace (delece a duplikace) hrají jednu z hlavních rolí v patogenezi mnoha geneticky podmíněných onemocnění člověka a mj. představují i častou příčinu mentálních retardací (MR). V posledních několika letech bylo potvrzeno, že se na patogenezi MR významně podílejí chromozomové abnormality s rozsahem pod detekční hranicí klasické cytogenetické analýzy. Tyto tzv. copy number variants (CNVs, odchylky v počtu kopií) jsou běžně detekovány pomocí různých celogenomových platforem s vysokou rozlišovací schopností. Při těchto celogenomových vyšetřeních dochází často k identifikaci nových mikrodelečních nebo mikroduplikačních syndromů nebo vzácně i k objevení či upřesnění kandidátních nebo přímo kauzálních genů pro již známé onemocnění.

MR se jako nevyléčitelné postižení objevuje u 2% nově narozených dětí. Vzhledem k tomu, že příčina tohoto onemocnění zůstává neobjasněna u více než 50% případů, je diagnostika MR a optimalizace jednotlivých laboratorních postupů jedním z nejčastějších zaměření genetického výzkumu. Právě díky rychlému rozvoji a zavádění nových technik molekulární cytogenetiky bylo zjištěno, že chromozomové aberace v subtelomerových oblastech jsou zodpovědné za postižení až u 5% případů idiopatické MR. Od roku 2002 patří k technikám umožňujícím odhalovat kryptické přestavby chromozomů i metoda MLPA.

V předložené studii byl pomocí techniky MLPA vyšetřen soubor 233 pacientů s idiopatickou MR či opožděným vývojem, diagnostikovaných na Oddělení lékařské genetiky v letech 2007-2010. Prostřednictvím kitů MLPA P036 a P070, navržených pro detekci přestaveb v subtelomerických oblastech všech lidských chromozomů, byly odhaleny chromozomové aberace u 14/233 případů, což odpovídá záchytu 6%. Celkově bylo touto technikou nalezeno, 21 subtelomerických změn u 14 pacientů, tyto abnormality byly následně ověřeny a podrobně charakterizovány metodami FISH a array-CGH.

Analýza a srovnání dostupných vzorků DNA pacientů a jejich rodičů prokázala, že čtyři aberace se objevily u pacientů de novo: dvě del(1)(p36) a dvě del(22)(p13). U šesti pacientů byly chromozomové abnormality zděděny po rodičích: dup(3)(q29), del(1)(p36) spojená s dup(12)(q24), dup(1)(p36) spojená s del(12)(q24), del(21)(q22) spojená s dup(11)(q24), dup(7)(q13) spojená s dup(19)(q13) a del(22)(q13), del(18)(p11). Ve čtyřech případech nebylo možné definovat původ aberací z důvodu chybějícího biologického materiálu: dup(4)(q32) spojená s del(7)(q35), dup(1)(p36), dup(17)(p13) spojená s del(22)(q13), del (16)(q24). V práci byla podrobně studována korelace nalezených genetických abnormalit s klinickými fenotypy.

Na základě zkušeností a dílčích výsledků byla navržena a optimalizována strategie co nejefektivnějšího vyšetřování pacientů s neobjasněnou MR na Oddělení lékařské genetiky.

Klasické cytogenetické vyšetření karyotypu (G-pruhování) zůstává prvním prováděným genetickým testem. MLPA zaujala stabilní místo při subtelomerickém a mikrodelečním skríninku. Technologií volby se v indikovaných případech stal celogenomový skrínink pomocí array-CGH. Cílená analýza pomocí metody FISH je s výhodou využívána při ověřování odhalených chromozomových změn během skríninku.

Naše výsledky potvrdily, že metoda MLPA a její využití v rutinní laboratorní diagnostice je schopno zvýšit záchyt kauzálních chromozomových aberací postihující subtelomerické oblasti chromozomů, které by jinak zůstaly nezachyceny a které mohou být příčinou MR či opožděného vývoje pacientů. Byla prokázána spolehlivost, účinnost a finanční efektivnost metody MLPA, která se tak stala rutinní diagnostickou laboratorní metodou využívanou na Oddělení lékařské genetiky Fakultní nemocnice Brno.

CONTENT

1 INTRODUCTION

1.1 Congenital chromosomal aberrations

1.1.1 Copy-number variation as a cause of genomic diseases

1.1.2 Distinguishing pathogenic and benign CNVs

1.1.3 New microdeletion/microduplication syndromes

1.2 Mental retardation and/or developmental delay (MR/DD)

1.2.1 Causes of mental retardation

1.2.2 Diagnosis, prognosis and treatment of patients with MR

1.3 Multiplex Ligation-dependent Probe Amplification (MLPA)

1.3.1 Principle, importance and utilization of MLPA

1.3.2 Different variants of MLPA

1.4 Investigation of patients with unexplained MR/DD

1.4.1 Cytogenetic and molecular cytogenetic techniques in examination of patients with MR/DD..... 27 1.4.2 Detection rate of chromosomal aberration using various technologies

1.4.3 Subtelomeric screening using MLPA

2 AIM OF STUDY

3 MATERIAL AND METHODS

3.1 Cytogenetic and molecular cytogenetic methods in used

4 RESULTS

4.1 Implementation of MLPA into the laboratory practice

4.2 The cohort of patients examined by subtelomeric screening

4.2.1 De novo submicroscopic aberrations

4.2.2 Familial submicroscopic aberrations

4.2.3 Aberrations with undetermined origin

4.3 Correlation genotype/phenotype in Cases 1-14

4.4 Diagnostic algorithm proposed for unexplained MR cases

5 DISCUSSION

6 CONCLUSION

7 REFERENCES

8 LIST OF SUPPLEMENTS

1 INTRODUCTION

1.1 Congenital chromosomal aberrations Chromosomal aberrations reflect an atypical number of chromosomes or a structural abnormality in the chromosomes. Congenital chromosomal rearrangements refer to those that are inherited from a parental carrier or occur de novo in the gametes. Chromosomal aberrations are proved to be common causes of mental retardation (MR). A cytogenetic imbalance is detected in about 0.6% of all newborn infants, 25% of all miscarriages and stillbirths, and 50–60% of first trimester miscarriages (Shaffer and Lupski, 2000). Most often, the recognized imbalance is caused by an aneuploidy. However, the most frequent constitutional aberrations are trisomies 13, 18, 21 and anomalies of sex chromosomes, other chromosomal structural abnormalities (duplication, deletion, inversion or translocation) could also cause a pathological phenotype (Schinzel 2001). The prevalence of the congenital structural chromosomal rearrangement in the general population is shown in Table 1.

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