Short-chain acyl-CoA dehydrogenase deficiency (SCADD) represents a rare autosomal recessive inborn metabolic disorder of mitochondrial β-oxidation of monocarboxylic acids. Clinical symptoms can vary from a severe life-threatening condition to an asymptomatic state, reported in the majority of cases. Since the expansion of newborn screenings, more than three hundred probands were admitted for molecular-genetic analysis, most selected because of elevated values of C4-acylcarnitine detected in newborn screenings in Slovakia. Searching for the principal genomic changes led us to the selection of sixty-two patients in whom the presence of sequence variants in the ACADS gene was analysed and correlated with the available biochemical and clinical data. Biochemical and molecular genetic tests were performed. Acylcarnitine profiles focused on an elevated level of C4-acylcarnitine, which was analysed via tandem mass spectrometry. Urinary organic acids, specifically a quantity of ethylmalonic acid, were determined by gas chromatography/mass spectrometry. The entire coding region of the ACADS gene was sequenced. A low-cost restriction fragment length polymorphism of PCR amplified fragments analysis (PCR-RFLP) of pathogenic variants was introduced and implemented for the molecular-genetic algorithm appropriate for the Slovak population. Our molecular genetic study was performed on sixty-two patients with a pathological biochemical pattern related to short-chain acyl-CoA dehydrogenase deficiency. In this cohort, we discovered a high occurrence of two rare pathogenic variants—the deletion c.310_312delGAG and the substitution c.1138C>T, with allelic frequencies of 64% and 31%, respectively. Up to 86% of investigated individuals belong to the Roma ethnic group. Analogous to other countries, SCADD is not included in the newborn screening programme. Based on the exceeded levels of the specific biomarker C4-acylcarnitine as well as ethylmalonic acid, we revealed a high prevalence of short-chain acyl-CoA dehydrogenase deficiency cases, confirmed by the findings of two rare pathogenic variants. A deletion c.310_312delGAG and c.1138C > T substitution in the ACADS gene appear with a high frequency in the Roma ethnic group of Slovakia. Due to the uncertainty of the pathogenicity and clinical consequences, it is important to follow up the morbidity and mortality in these patients over time and evaluate SCADD in relation to clinical outcomes and preventive healthcare recommendations.

Renal hypouricemia (RHUC) is caused by an inherited defect in the main reabsorption system of uric acid, SLC22A12 (URAT1) and SLC2A9 (GLUT9). RHUC is characterized by a decreased serum uric acid concentration and an increase in its excreted fraction. Patients suffer from hypouricemia, hyperuricosuria, urolithiasis, and even acute kidney injury. We report clinical, biochemical, and genetic findings in a cohort recruited from the Košice region of Slovakia consisting of 27 subjects with hypouricemia and relatives from 11 families, 10 of whom were of Roma ethnicity. We amplified, directly sequenced, and analyzed all coding regions and exon–intron boundaries of the SLC22A12 and SLC2A9 genes. Sequence analysis identified dysfunctional variants c.1245_1253del and c.1400C>T in the SLC22A12 gene, but no other causal allelic variants were found. One heterozygote and one homozygote for c.1245_1253del, nine heterozygotes and one homozygote for c.1400C>T, and two compound heterozygotes for c.1400C>T and c.1245_1253del were found in a total of 14 subjects. Our result confirms the prevalence of dysfunctional URAT1 variants in Roma subjects based on analyses in Slovak, Czech, and Spanish cohorts, and for the first time in a Macedonian Roma cohort. Although RHUC1 is a rare inherited disease, the frequency of URAT1-associated variants indicates that this disease is underdiagnosed. Our findings illustrate that there are common dysfunctional URAT1 allelic variants in the general Roma population that should be routinely considered in clinical practice as part of the diagnosis of Roma patients with hypouricemia and hyperuricosuria exhibiting clinical signs such as urolithiasis, nephrolithiasis, and acute kidney injury.

Wilson disease (WD) is an autosomal recessive disorder of copper transport caused by bi‐allelic pathogenic variants in the ATPase copper transporting beta gene ( ATP7B ). Results of standard genetic diagnostics remain inconclusive in 3%–20% of WD patients in part due to problematic assessment of variants of unknown or conflicting pathogenicity (synonymous variants included). Correct interpretation of potential effects of such variants can be substantially enhanced by RNA analyses. This strategy is, however, of limited utility in WD patients because of predominant liver expression of ATP7B . To avoid invasive bioptic liver collection and increase WD diagnostic yields, we searched for a surrogate tissue sample and identified profiles of ATP7B transcripts in nasopharyngeal swabs that were comparable to liver. Amplicons spanning ATP7B Exons 3–21 were prepared from the swab material and analysed by long‐read nanopore sequencing to enable the detection of splicing changes and variant phasing. Diagnostic utility of this novel in vivo methodology was demonstrated by characterization of mRNA splicing abnormalities caused by synonymous ATP7B variants c.1488C>T (p.(Gly496=)), c.2241C>T (p.(Ile747=)), c.2292C>T (p.(Phe764=)), and a nonsense variant c.2336G>A (p.(Trp779Ter)) in four WD patients, who were not genetically resolved by standard techniques. Nasopharyngeal swab sampling is minimally invasive and allows effective analyses of mRNA to detect and/or validate effects of ATP7B variants in WD patients. Conclusive genetic diagnosis attained by this novel technique may facilitate family counselling and substantiate initiation of copper‐chelation therapy in presymptomatic individuals.

Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an autosomal recessive defect of L-isoleucine catabolism caused by pathogenic variants in the ACADSB gene. While early reports described neurological symptoms, expanded newborn screening cohorts have revealed predominantly asymptomatic individuals, raising questions regarding the clinical significance and optimal management. We performed an evaluation of three probands with a biochemical profile consistent with SBCADD identified in a Central European country. Acylcarnitines were measured by liquid chromatography with tandem mass spectrometry, urinary organic acids by gas chromatography with mass spectrometry, and ACADSB was sequenced. Clinical data were collected from regional paediatric follow-up. Three individuals with a biochemical profile consistent with SBCADD were identified in Slovakia. Two were detected through newborn screening, whereas one was diagnosed at 5 years of age. All three showed elevated C5-acylcarnitine; the C5/C8 ratio was increased in two patients, while in the third it remained within the reference range despite persistently elevated C5. Urinary 2-methylbutyrylglycine and 2-ethyl-3-hydroxypropionate were elevated in all cases and showed intra-individual variability; in one sample, 2-methylbutyrylglycine remained increased while 2-ethyl-3-hydroxypropionate had normalised. Two patients carried pathogenic ACADSB variants (one homozygous splice-site variant c.303 + 1G > A, one compound heterozygous for p.Thr148Ile and p.Glu387Lys), whereas genetic testing was not performed in the third case. Over the available follow-up period, no episodes of metabolic decompensation were observed; two patients remained clinically well, and one patient had autism spectrum disorder. Free carnitine concentrations were within the reference range in all probands. Our findings support SBCADD as a primarily biochemical phenotype with a largely benign course, while illustrating variability of urinary biomarkers and C5/C8 ratios. Accurate differentiation from isovaleric acidemia and careful integration of biochemical, genetic and clinical data remain essential.