👤 Elżbieta Jurkiewicz

Whole-exome sequencing (WES) has led to an exponential increase in identification of causative variants in mitochondrial disorders (MD). We performed WES in 113 MD suspected patients from Polish paediatric reference centre, in whom routine testing failed to identify a molecular defect. WES was performed using TruSeqExome enrichment, followed by variant prioritization, validation by Sanger sequencing, and segregation with the disease phenotype in the family. Likely causative mutations were identified in 67 (59.3 %) patients; these included variants in mtDNA (6 patients) and nDNA: X-linked (9 patients), autosomal dominant (5 patients), and autosomal recessive (47 patients, 11 homozygotes). Novel variants accounted for 50.5 % (50/99) of all detected changes. In 47 patients, changes in 31 MD-related genes (ACAD9, ADCK3, AIFM1, CLPB, COX10, DLD, EARS2, FBXL4, MTATP6, MTFMT, MTND1, MTND3, MTND5, NAXE, NDUFS6, NDUFS7, NDUFV1, OPA1, PARS2, PC, PDHA1, POLG, RARS2, RRM2B, SCO2, SERAC1, SLC19A3, SLC25A12, TAZ, TMEM126B, VARS2) were identified. The ACAD9, CLPB, FBXL4, PDHA1 genes recurred more than twice suggesting higher general/ethnic prevalence. In 19 cases, variants in 18 non-MD related genes (ADAR, CACNA1A, CDKL5, CLN3, CPS1, DMD, DYSF, GBE1, GFAP, HSD17B4, MECP2, MYBPC3, PEX5, PGAP2, PIGN, PRF1, SBDS, SCN2A) were found. The percentage of positive WES results rose gradually with increasing probability of MD according to the Mitochondrial Disease Criteria (MDC) scale (from 36 to 90 % for low and high probability, respectively). The percentage of detected MD-related genes compared with non MD-related genes also grew with the increasing MD likelihood (from 20 to 97 %). Molecular diagnosis was established in 30/47 (63.8 %) neonates and in 17/28 (60.7 %) patients with basal ganglia involvement. Mutations in CLPB, SERAC1, TAZ genes were identified in neonates with 3-methylglutaconic aciduria (3-MGA) as a discriminative feature. New MD-related candidate gene (NDUFB8) is under verification. We suggest WES rather than targeted NGS as the method of choice in diagnostics of MD in children, including neonates with 3-MGA aciduria, who died without determination of disease cause and with limited availability of laboratory data. There is a strong correlation between the degree of MD diagnosis by WES and MD likelihood expressed by the MDC scale. Show less

Genotype-phenotype associations were studied in 517 subjects clinically affected by classical neuronal ceroid lipofuscinosis (NCL). Genetic loci CLN1-3 were analyzed in regard to age of onset, initial neurological symptoms, and electron microscope (EM) profiles. The most common initial symptom leading to a clinical evaluation was developmental delay (30%) in NCL1, seizures (42.4%) in NCL2, and vision problems (53.5%) in NCL3. Eighty-two percent of NCL1 cases had granular osmiophilic deposits (GRODs) or mixed-GROD-containing EM profiles; 94% of NCL2 cases had curvilinear (CV) or mixed-CV-containing profiles; and 91% of NCL3 had fingerprint (FP) or mixed-FP-containing profiles. The mixed-type EM profile was found in approximately one-third of the NCL cases. DNA mutations within a specific CLN gene were further correlated with NCL phenotypes. Seizures were noticed to associate with common mutations 523G>A and 636C>T of CLN2 in NCL2 but not with common mutations 223G>A and 451C>T of CLN1 in NCL1. Vision loss was the initial symptom in all types of mutations in NCL3. Surprisingly, our data showed that the age of onset was atypical in 51.3% of NCL1 (infantile form) cases, 19.7% of NCL2 (late-infantile form) cases, and 42.8% of NCL3 (juvenile form) cases. Our data provide an overall picture regarding the clinical recognition of classical childhood NCLs. This may assist in the prediction and genetic identification of NCL1-3 via their characteristic clinical features. Show less

Late-infantile neuronal ceroid lipofuscinosis (LINCL), an autosomal recessively inherited lysosomal storage disorder characterized by autofluorescent inclusions and rapid progression of neurodegeneration, is due to CLN2 gene mutations. However, CLN2 mutation analysis has failed to identify some clinically diagnosed "late-infantile" NCL cases. This study was conducted to further characterize genetic heterogeneity in families affected by LINCL. DNA mutations in the CLN1, CLN2, and CLN3 genes that underlie INCL (infantile NCL), LINCL, and JNCL (juvenile NCL), respectively, were studied with molecular analyses. A total of 252 families affected by childhood NCL were studied. Of 109 families clinically diagnosed as having LINCL, 3 were determined to have either INCL or JNCL by identification of mutation(s) in CLN1 or CLN3. Six families diagnosed initially as having JNCL were found to have LINCL based on the finding of mutations in the CLN2 gene. In addition, several novel mutations were identified. Clinical and genetic heterogeneity of LINCL was demonstrated in nine LINCL families studied. Show less

The neuronal ceroid lipofuscinoses (NCLs) are a large group of autosomal recessive lysosomal storage disorders with both enzymatic deficiency and structural protein dysfunction. Three typical forms, the infantile (INCL), late-infantile (LINCL), and juvenile (JNCL), are among the most common childhood-onset neurodegenerative disorders. They result from mutations on genes CLN1, CLN2, and CLN3, respectively. We determined that the mutations 223A --> G and 451C --> T in CLN1, T523-1G --> C, and 636 C --> T in CLN2, and deletion of a 1.02-kb genomic fragment in CLN3 are the five common mutations for NCL. To offer clinical genetic testing for the NCLs, we have developed simple and quick PCR-based molecular tests for detecting INCL-, LINCL-, and JNCL-affected individuals from 180 NCL families (27 INCL, 76 LINCL, and 77 JNCL). The sensitivity of testing to detect NCL patients among clinically suspected individuals was determined to be 78% (21/27) for INCL, 66% (54/76) for LINCL, and 75% (58/77) for JNCL. When molecular screening for carriers was conducted among the normal siblings or parents of the probands, we identified two carriers out of three individuals tested for INCL, 20/56 (35.7%) carriers for LINCL, and 48/106 (45.3%) carriers for JNCL families. In addition, 5% (9/180) of NCL patients revealed genetic heterogeneity and were reclassified. Seven patients previously diagnosed as having JNCL were now found to carry mutations of CLN2 (5/7) or CLN1 (2/7) and 2 with late-infantile onsets were identified as carrying mutations of CLN1. Our data demonstrate the importance of DNA testing to detect accurately both affected individuals and carriers in NCL families. Show less

The lysosomal storage of lipofuscins is the common pathological feature that characterizes the infantile, late-infantile, juvenile (Batten's disease), and Finnish-variant neuronal ceroid lipofuscinosis (INCL, LINCL, JNCL and FNCL), which are due to mutations in the genes CLN1, CLN2, CLN3, and CLN5, respectively. The CLN1 and CLN2 genes encode lysosomal enzymes, but the CLN3 and CLN5 genes encode membrane-spanning proteins. Why deficiencies of lysosomal enzymes and membrane-spanning proteins produce similar clinical phenotypes and pathological changes is still unanswered. We hypothesize that CLN-encoded proteins may comprise a functional pathogenic pathway, in which protein associations may play important roles. To test this hypothesis, we studied protein-protein interactions among the CLN1-, CLN2-, and CLN3-encoded proteins using a yeast two-hybrid system. Our results provided no evidence that CLN-encoded proteins interact with each other. This suggests there may be unidentified components in NCL pathogenesis. Show less