Struktura segmentalne duplikacije 5q13.2 kao modifikator fenotipa spinalne mišićne atrofije i amiotrofične lateralne skleroze

Abstract

Spinalna mišićna atrofija (SMA) i amiotrofična lateralna skleroza (ALS) su bolesti motoneurona. SMA je najčešći monogenski uzročnik smrtnosti dece, dok je ALS adultna, neizlečiva bolest sa vrlo progresivnim tokom. SMA je uzrokovana homozigotnim odsustvom gena SMN1 u telomernom delu segmentalne duplikacije 5q13.2, dok se u njenom centromernom delu nalazi gotovo istovetan gen SMN2 koji daje ~10% funkcionalnog proteina SMN. Klinička prezentacija SMA je izuzetno varijabilna i kreće se od najtežeg oblika kod novorođenčadi (tip I) do blagog oblika sa adultnim početkom (tip IV). Homogenost uzročne mutacije i izražena fenotipska heterogenost ukazuju na postojanje genetičkih, epigenetičkih i sredinskih faktora koji modifikuju fenotip SMA. Visoka stopa nejednakih hromozomskih rearanžmana u segmentalnoj duplikaciji 5q13.2 dovodi do varijacija u broju kopija i strukturnih promena ne samo gena SMN, već i drugih prisutnih gena, poput SERF1 i NAIP, i one mogu uticati na tok i težinu bolesti. Dosadašnji podaci ukazuju na to da bi jednim delom uzrok fenotipskih razlika kod SMA mogao biti različit stepen metilacije promotora SMN2. Sporadična ALS (SALS) čini oko 90% svih slučajeva ALS i njena etiologija nije razjašnjena. Iako se genetička osnova, vreme pojave simptoma i tok SMA i SALS razlikuju, usled sličnosti patoloških promena i učešća proteina SMN i proteina asociranih sa ALS u zajedničkim molekularnim putevima, među genima kandidatima za nastanak i progresiju SALS velika pažnja usmerena je upravo na gene SMN. Cilj ove doktorske disertacije bio je ispitivanje genomske strukture, prirode rearanžmana i epigenetičkih oznaka u segmentalnoj duplikaciji 5q13.2 kod SMA bolesnika. Takođe, cilj je bio ispitivanje asocijacije broja kopija gena SMN1, SMN2, SERF1 i NAIP sa fenotipom dečije SMA, rizikom za nastanak SALS i preživljavanjem SALS bolesnika. U grupi od 99 SMA bolesnika i 122 roditelja bolesnika metodom umnožavanja većeg broja ligiranih proba (MLPA) analizirana su 442 hromozoma i identifikovano je 20 različitih alela telomernog dela duplikacije 5q13.2. Između broja kopija gena SMN2, SERF1 i NAIP i tipa dečije SMA uočena je statistički značajna obrnuto srazmerna korelacija (Spirmanov test korelacije, SMN2 p=2,2e-16, SERF1 p=4,264e-10, NAIP p=2,722e-8). Generalizovanim linearnim modelom i selekcijom unazad, počevši od punog modela koji je obuhvatio broj kopija SMN2, SERF1 i NAIP i njihove dvostruke interakcije, dobijen je najbolji minimalni model koji objašnjava fenotipsku varijabilnost dečije SMA i obuhvata broj kopija SMN2 (p<2e-16) i SERF1 (p<2e-16) i njihov udruženi efekat (p=0,026)...

Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are motor neurone diseases. SMA is the most frequent monogenic cause of infant mortality, whereas ALS is an incurable late-onset disease with an inexorably progressive course. SMA is caused by homozygous absence of the SMN1 gene located in the telomeric part of the 5q13.2 segmental duplication, while in its centromeric part resides a nearly identical copy named SMN2 producing only ~10% of functional SMN protein. Clinical presentation of SMA is extremely variable ranging from a severe infantile form (type I) to mild adult-onset form (type IV). Homogeneity of the disease-causing mutation and extensive phenotypic variability clearly indicate the existence of additional genetic, epigenetic and environmental factors modulating SMA phenotype. High rate of unequal crossing over in 5q13.2 segmental duplication results in copy number variations (CNVs) and complex structural rearrangements of the two SMN genes and other genes residing in the same region, such as SERF1 and NAIP, modulating disease progression and severity. Existing data suggest that, to some extent, phenotypic discrepancies in SMA might be due to differences in methylation level of the SMN2 promoter. Sporadic ALS (SALS) comprises ~90% of all ALS cases and has an unknown aetiology. Even though aetiology, age at onset and progression may differ between the diseases, due to similarities between ALS and SMA pathologies and the involvement of SMN and some ALS-associated proteins in common molecular pathways, SMN1 and SMN2 genes were repeatedly studied as SALS susceptibility genes. The aim of this doctoral thesis was to reconstruct genomic structure, the nature of rearrangements and epigenetic marks of the 5q.13.2 segmental duplication in SMA patients from Serbia. Furthermore, the aim was to investigate if copy numbers of the SMN1, SMN2, SERF1 and NAIP genes are associated with SMA type, susceptibility to SALS and survival in SALS patients from Serbia. In a group of 99 SMA patients and 122 patients’ parents 442 chromosomes were analysed by multiplex ligation-dependent probe amplification (MLPA) and 20 different alleles of the 5q13.2 telomeric part were observed. Statistically significant inverse correlation was found between SMN2, SERF1 and NAIP CNVs and SMA type (Spearman rank test, SMN2 p=2.2e-16, SERF1 p=4.264e-10, NAIP p=2.722e-8). Generalised linear model and backward selection, starting with a full model including the SMN2, SERF1 and NAIP copy number and their two-way interactions, revealed that the best minimal model explaining phenotypic variation in childhood-onset SMA with the smallest set of variables included the SMN2 (p<2e-16) and SERF1 (p<2e-16) copy number and their joint effect (p=0.02628)...