ObjectiveReaching a genetic diagnosis of mitochondrial disorders (MDs) is challenging due to their broad phenotypic and genotypic heterogeneity. However, there is growing evidence that the use of whole exome sequencing (WES) for diagnosing patients with a clinical suspicion of an MD is effective (39–60%). We aimed to study the effectiveness of WES in clinical practice in Estonia, in patients with an unsolved, but suspected MD. We also show our first results of mtDNA analysis obtained from standard WES reads.MethodsRetrospective cases were selected from a database of 181 patients whose fibroblast cell cultures had been stored from 2003 to 2013. Prospective cases were selected during the period of 2014–2016 from patients referred to a clinical geneticist in whom an MD was suspected. We scored each patient according to the mitochondrial disease criteria (MDC) (Morava et al., 2006) after re-evaluation of their clinical data, and then performed WES analysis.ResultsA total of 28 patients were selected to the study group. A disease-causing variant was found in 16 patients (57%) using WES. An MD was diagnosed in four patients (14%), with variants in the SLC25A4, POLG, SPATA5, and NDUFB11 genes. Other variants found were associated with a neuromuscular disease (SMN1, MYH2, and LMNA genes), neurodegenerative disorder (TSPOAP1, CACNA1A, ALS2, and SCN2A genes), multisystemic disease (EPG5, NKX1–2, ATRX, and ABCC6 genes), and one in an isolated cardiomyopathy causing gene (MYBPC3). The mtDNA point mutation was found in the MT-ATP6 gene of one patient upon mtDNA analysis.ConclusionsThe diagnostic yield of WES in our cohort was 57%, proving to be a very good effectiveness. However, MDs were found in only 14% of the patients. We suggest WES analysis as a first-tier method in clinical genetic practice for children with any multisystem, neurological, and/or neuromuscular problem, as nuclear DNA variants are more common in children with MDs; a large number of patients harbor disease-causing variants in genes other than the mitochondria-related ones, and the clinical presentation might not always point towards an MD. We have also successfully conducted analysis of mtDNA from standard WES reads, providing further evidence that this method could be routinely used in the future.

The urinary creatine:creatinine (Cr:Crn) ratio was measured in males from 49 families with a family history compatible with X-linked mental retardation (XLMR) in order to estimate the prevalence of SLC6A8 deficiency in Estonia. We identified 11 boys from 9 families with an increased urinary Cr:Crn ratio (18%). In three related boys, a hemizygous missense mutation (c.1271G>A; p.Gly424Asp) was identified. Their mother was heterozygous for the same mutation. Although many missense mutations have been described, the p.Gly424Asp mutation has not been previously reported. The clinical expression varied widely among affected males of this family. Patients 1 and 3 had relatively mild clinical expression (mild mental retardation (MR) and attention deficit disorder), but patient 2 had all typical clinical signs of SLC6A8 defect such as moderate MR, autistic features, expressive dysphasia and epilepsy. Among our patients, we saw significant problems in speech and language development combined with attention and behavioural difficulties. The number of false-positive biochemical results with increased urinary Cr:Crn ratio was higher (18%) in our study than in previous reports (1.8–10%). We therefore suggest that repeated biochemical testing should be performed before DNA sequencing analysis. Our study suggests that 2% (95% confidence limits: 0.05–11.1%) of this Estonian XLMR panel are due to mutations in the SLC6A8, which is similar to the prevalence reported in other populations. We therefore conclude that creatine transporter deficiency is a relatively common genetic disorder in males with sporadic or familiar MR and diagnostic screening of them should always include screening for SLC6A8 deficiency.

The HLA-Cw1 and -Cw2 genes were identified in a genomic library and their products characterized by biochemical methods. The HLA-Cw1 and -Cw2 genes, upon transfection in mouse L cells, give rise to class I antigen heavy chains that associate with neither mouse nor human beta-2 microglobulin. They are indistinguishable in isoelectric point from polypeptides identified as HLA-Cw1 and -Cw2 in human cells. The nucleotide sequence of HLA-Cw1 and -Cw2 and their comparison with HLA-Cw3, the only other known HLA-C sequence, reveal a characteristic pattern of locus-specific amino acids. A comparison of 13 different human class I primary structures leads us to speculate that the most variable region in HLA class I antigens, positions 61-83, could assume an alpha helical structure of critical importance for class I antigen function. The locus specificity and the higher degree of intralocus conservation in the COOH-terminal region, especially in the transmembrane and cytoplasmic domains, must reflect evolutionary ancestry rather than positive selection. In view of the pattern and types of substitutions observed for HLA-C locus products, their function as immune response gene products is questioned.

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The nucleotide sequences of the human class I major histocompatibility complex genes HLA‐B27k and HLA‐B27w have been determined. They differ by only four nucleotides over a stretch of 14 bp in exon 2, resulting in three amino acid exchanges at positions 77 (Asp to Asn), 80 (Thr to Ile) and 81 (Leu to Ala). The distribution of these nucleotide substitutions suggests a gene conversion‐like event responsible for the generation of these HLA‐B27 subtypes. The mechanisms underlying the generation of new polymorphic variants in man are therefore probably identical to the gene conversion‐like events postulated in the generation of H‐2Kbm class I mutants in the mouse.