| Abstract | Introduction and aim. Childhood-onset systemic lupus erythematosus (cSLE) and IgA
vasculitis (IgAV) are two complex autoimmune diseases with etiopathogenesis which are not
fully understood. This research aimed to identify novel and rare gene variants using wholeexome
sequencing (WES) in patients with cSLE and IgAV which may contribute to the
etiopathogenesis of these diseases, and to expand existing genetic databases.
----- Subjects and methods. WES was performed on 17 "trio" groups containing a proband case
with cSLE and parents (including other informative family members) and on 3 "trios"
containing a proband case with IgAV, and parents with severe, atypical clinical features,
syndromic characteristics, early onset of the disease, resistance to conventional therapy and/or
a family pattern of occurrence. After completion of WES, data analysis and the identification
of all the genetic variants of interest, the presence of an interferon gene expression signature in
peripheral blood mononuclear cell samples was investigated on one patient.
----- Results. After performing WES and analysis of gene variants, novel and/or rare variants were
detected in 8 patients. Among them, there were 7 patients diagnosed with cSLE and 1 patient
with IgAV, while 1 patient was diagnosed with cSLE and IgAV in a different period. A total
number of 20 variants were prioritised for more detailed analysis. WES analysis yielded a new
pathogenic variant in the histone-lysine N-methyltransferase 2D gene (KMT2D),
NM_003482.3:c.8626delC, predicted to truncate the protein (p.Gln2876Serfs*34) resulting in
KMT2D loss of function, explaining a previously unrecognised syndrome which features in a
patient with Kabuki syndrome and cSLE. In addition, likely pathogenic variants were identified
in the adenosine deaminase acting on the RNA gene (ADAR1), NM_001111.3:c.2815A>G,
predicted to encode the protein (p.Ile939Val) in a patient with dysmorphic features, skin
pigmentation changes and cSLE, only partially explaining the very complex phenotype, as well
as in the B lymphocyte kinase gene (BLK), NM_001715.2;c.211G>A, predicted to encode
(p.Ala71Thr) in a patient with severe cSLE and multisystemic involvement with the same
disease in her mother which is worth further functional analysis. Among the number of variants
of uncertain significance (VUS) in patients with cSLE and IgAV, the most promising, in terms
of contribution to the development of the disease and the impact on the clinical picture, are
recombination activating 2, RAG2 (NM_000536.3:c.1393A>G) predicted to encode
(p.The465Ala), tyrosine-protein kinase 2, TYK2 (NM_003331.4:c.2492A>T) predicted to
encode (p.Asp810Val), B-cell lymphoma 2 associated antagonist of cell death, BAD
(NM_004322.3:c.462G>C) predicted to encode (p.Trp154Cys), and DExH-box helicase 58,
DHX58 (NM_024119.2:c.560A>G) predicted to encode (p.Gln187Arg), making them
candidates for further function analyses. ----- Conclusion. In the new epoch of personalised medicine, implementation of next generation
sequencing has improved the diagnostics and treatment of patients with autoimmune diseases
Nevertheless, such quantities of data raised the problem of interpretation of genetic variants and
their use for therapeutic purposes. Undeniably, pathogenic variants often represent only a small
percentage of all the variants reported, while there is growing number of variants which we are
still not able to clearly define and use in a clinical context, such as VUS, which limit the clinical
utility of genetic information. This has to prompt the scientific community to develop methods
to properly categorise VUS and escalate the amount of practicable information from next
generation sequencing. |
| Abstract (croatian) | Uvod i cilj. Sistemski eritemski lupus koji započinje u dječjoj dobi (cSLE) i IgA vaskulitis
(IgAV) dvije su kompleksne autoimunosne bolesti još uvijek nepotpuno razjašnjene
etiopatogeneze. Cilj ovog istraživanja bio je primjenom sekvenciranja cijelog egzoma (WES)
u bolesnika cSLE-om i IgAV-om identificirati nove i rijetke genske varijante koje mogu
doprinijeti etiopatogenezi ovih bolesti i proširiti postojeće genske baze podataka.
----- Ispitanici i metode. Skupina od 17 bolesnika s cSLE-om kojima su bila pridružena i oba
roditelja (uključujući i ostale informativne članove obitelji) te 3 bolesnika s IgAV-om, također
s pridružena oba roditelja, s teškom ili atipičnom kliničkom slikom, sindromskim obilježjima,
ranim početkom bolesti, rezistencijom na konvencionalnu terapiju i/ili obiteljskom pojavnošću
bolesti, podvrgnuta je WES-u. Nakon kompletiranja WES-a, analize podataka i identifikacije
svih genskih varijanti od interesa, u jednog je bolesnika učinjeno određivanje interferonskog
genskog potpisa iz mononuklearnih stanica periferne krvi.
----- Rezultati. Nakon učinjenog WES-a i analize genskih varijanti, nove i/ili rijetke varijante
detektirane su u 8 bolesnika. Među njima bilo je 7 bolesnika s cSLE-om i 1 bolesnik s IgAVom,
a 1 bolesnik imao je cSLE i IgAV, ali u različitom periodu. Odabrano je 20 varijanti koje
su podvrgnute detaljnoj analizi. Primjenom WES-a otkrivena je nova patogena varijanta u genu
za histon-lizin N-metiltransferazu 2D (KMT2D), NM_003482.3:c.8626delC, za koju se
predviđa da kodira trunkirani protein (p.Gln2876Serfs*34), čime dovodi do gubitka funkcije
KMT2D, što je pomoglo objasniti prethodno neprepoznata sindromska obilježja u bolesnice s
Kabukijevim sindromom i cSLE-om. Također su pronađene vjerojatno patogene varijante u
genu za adenozin deaminazu koji djeluje na RNA (ADAR1), NM_001111.3:c.2815A>G, za
koju se predviđa da kodira protein (p.Ile939Val), u bolesnice sa sindromskim obilježjima,
promjenama pigmentacije i cSLE-om, kojom je samo djelomično moguće objasniti njezin
kompleksni fenotip, te u genu za B limfocitnu kinazu (BLK), NM_001715.2;c.211G>A, za koju
se previđa da kodira (p.Ala71Thr), u bolesnice s teškim cSLE-om i multiorganskim
manifestacijama te istom bolešću u majke, a koje bi bilo korisno podvrgnuti daljnjim
funkcijskim analizama. Među brojnim varijantama nesigurna značenja (VUS) u bolesnika s
cSLE-om i IgAV-om, najperspektivnijima, u smislu mogućeg doprinosa u nastanku bolesti i
utjecaja na kliničku sliku, pokazale su se varijante u genu za aktivaciju rekombinacije 2, RAG2
(NM_000536.3:c.1393A>G) za koju se predviđa da kodira (p.The465Ala), u genu za tirozinsku
proteinsku kinazu 2, TYK2 (NM_003331.4:c.2492A>T), za koju se predviđa da kodira
(p.Asp810Val), u genu za B-stanični limfom 2 agonist stanične smrti, BAD
(NM_004322.3:c.462G>C), za koju se predviđa da kodira (p.Trp154Cys), te u genu za DExHbox
helikazu 58, DHX58 (NM_024119.2:c.560A>G), za koju se predviđa da kodira
(p.Gln187Arg) pa su ove varijante kandidati za daljnje funkcijske analize. ----- 201
Zaključak. U novoj epohi personalizirane medicine implementacijom sekvenciranja sljedeće
generacije došlo je do poboljšanja dijagnostike i liječenja bolesnika s autoimunosnim
bolestima. Međutim, tolika količina podataka otvorila je problem interpretacije genskih
varijanti i njihove primjene u terapijske svrhe. Nedvojbeno, patogene varijante često
predstavljaju samo mali postotak svih pronađenih varijanti dok raste broj varijanti koje još
uvijek ne možemo jasno definirati i primijeniti u kliničkom kontekstu, kao što su VUS, što
ograničava kliničku primjenjivost genskih informacija. Ovo bi trebalo potaknuti znanstvenu
zajednicu na razvoj metoda kojima bi se na odgovarajući način kategorizirale VUS varijante i
povećala količina praktičnih informacija koje možemo dobiti primjenom sekvenciranja sljedeće
generacije. |
