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.

Idiopathic infantile hypercalcemia (IIH) is a mineral metabolism disorder characterized by severe hypercalcemia, failure to thrive, vomiting, dehydration, and nephrocalcinosis. The periodical increase in incidence of IIH, which occurred in the twentieth century in the United Kingdom, Poland, and West Germany, turned out to be a side effect of rickets over-prophylaxis. It was recently discovered that the condition is linked to two genes, CYP24A1 and SLC34A1. The aim of the study was to search for pathogenic variants of the genes in adult persons who were shortlisted in infancy as IIH caused by “hypersensitivity to vit. D”. All persons were found to carry mutations in CYP24A1 or SLC34A1, nine and two persons respectively. The changes were biallelic, with one exception. Incidence of IIH in Polish population estimated on the basis of allele frequency of recurrent p.R396W CYP24A1 variant, is 1:32,465 births. It indicates that at least a thousand homozygotes and compound heterozygotes with risk of IIH live in the country. Differences in mechanism of developing hypercalcemia indicate that its prevention may vary in both IIH defects. Theoretically, vit. D restriction is a first indication for CYP24A1 defect (which disturbs 1,25(OH)₂D degradation) and phosphate supplementation for SLC34A1 defect (which impairs renal phosphate transport). In conclusion, we suggest that molecular testing for CYP24A1 and SLC34A1 mutations should be performed in each case of idiopathic hypercalcemia/hypercalciuria, both in children and adults, to determine the proper way for acute treatment and complications prevention.

Pyruvate dehydrogenase complex (PDHc) defect is a well-known cause of mitochondrial disorders (MD) with at least six responsible genes (PDHA1, PDHB, DLAT, DLD, PDHX, PDP1). The aim of this work was to assess the diagnostic value of biochemical methods in recognition of PDHc defect in Polish patients with suspicion of MD. In the first step, Western blot of the E1α subunit was performed on 86 archive muscle bioptates with suspicion of MD. In the second step, Sanger PDHA1 sequencing was performed in 21 cases with low E1α expression. In the third step, 7 patients with negative results of PDHA1 sequencing were subjected to whole-exome sequencing (WES). This protocol revealed 4 patients with PDHA1 and one with DLD mutations. Four additional probands were diagnosed outside the protocol (WES or Sanger sequencing). The molecular characterization of PDHc defect was conducted in a total of 9 probands: 5 according to and 4 off the protocol. Additionally, two affected relatives were recognized by a family study. Altogether we identified seven different PDHA1 changes, including two novel variants [c.464T > C (p.Met155Thr) and c.856_859dupACTT (p.Arg288Leufs*10)] and one DLD variant. The lactate response to glucose load in the PDHA1 subset was compared to a subset of non PDHc-related MD. Opposite responses were observed, with an increase of 23% and decrease of 27%, respectively. The results show that determining lactate response to glucose load and muscle E1α expression may contribute to distinguishing PDHc-related and other MD, however, WES is becoming the method of choice for MD diagnostics.

Glycogen synthase deficiency (glycogen storage disease 0 - GSD 0) caused by mutations in the GYS2 gene is characterized by a lack of glycogen synthesis in the liver. It is a rare condition of disturbed glycogen homeostasis in the liver with less than 30 cases reported in the literature so far. We report a 9 year old boy diagnosed with GSD 0 due to the newly identified, highly pathogenic homozygous mutation: NM_021957.3:p.Phe574Leu/c.1720T > C in ex. 14. A random, asymptomatic hypoglycemia with ketonuria was found in this patient at the age of 7. His developmental parameters were within normal ranges. Oral glucose tolerance test showed normal baseline blood levels of glucose, insulin and lactate, and their increase following glucose intake. Eight-hour fasting plasma glucose test, revealed glucose blood level of 34 mg/dl with no clinical symptoms. The results of these tests suggested GSD 0. Molecular analysis of the GYS2 gene was not feasible, but this particular gene was included in the panel of hypoglycemia of whole exome sequencing (WES) which was at our disposal.

Background: Alport syndrome (AS) is a clinically and genetically heterogeneous glomerulopathy resulting from pathogenic variants in COL4A3, COL4A4, and COL4A5. Genetic diagnosis is increasingly being conducted using next-generation sequencing (NGS). Methods: Within eight years, we examined a group of 247 Polish individuals and found in total 138 unrelated probands suspected with AS based on clinical course, laboratory findings, and/or family history, as well as the total of 109 family members. We applied a targeted NGS panel to identify the genetic spectrum of AS. Known and novel variants were revealed, and detailed evaluation was performed according to ACMG/AMP guidelines to classify them as pathogenic/likely pathogenic/VUS changes. Identified genotypes were compared with clinical manifestations: hematuria, proteinuria, chronic kidney disease, sensorineural hearing impairment, ocular abnormalities, and hypertension. Results: The molecular background was established in 109/138 probands. Overall, 79 different COL4A3-COL4A5 changes (56 known and 23 novel) were revealed. About 97% were SNVs, and only two COL4A5 CNVs were identified. In total, 11 recurrent COL4A3-COL4A5 variants were observed, including the most frequent COL4A5:p.Gly624Asp, accounting for 31% of X-linked AS. Conclusions: The use of NGS panel has shown considerable promise in the field of AS, increasing diagnostic rate to 79% and reducing time to diagnosis. The phenotype-driven gene panel, specific for genetic diseases in the pediatric population, is an affordable alternative to WGS and WES, offering comparable diagnostic efficacy and supporting its implementation as a first-line genetic test in rare diseases, including AS. Based on the obtained genotype–phenotype correlation, we assessed that NGS allows us to avoid invasive renal biopsy in AS diagnosis. It provides AS confirmation/exclusion, atypical AS identification, symptomatic/asymptomatic monoallelic COL4A3-COL4A5 carrier (especially COL4A5 females) determination, and inheritance pattern establishment. AS diagnosis confirmation enables clinical course prediction and is crucial for the early introduction of renoprotective treatment with renin–angiotensin–aldosterone system blockade, aimed at slowing the disease progression and estimating the risk in family members, which is important for genetic counselling.