Alkaptonuria (AKU) is a rare metabolic disorder caused by a deficient enzyme in the tyrosine degradation pathway, homogentisate 1,2-dioxygenase (HGD). In 172 AKU patients from 39 countries, we identified 28 novel variants of the HGD gene, which include three larger genomic deletions within this gene discovered via self-designed multiplex ligationdependent probe amplification (MLPA) probes. In addition, using a reporter minigene assay, we provide evidence that three of eight tested variants potentially affecting splicing cause exon skipping or cryptic splice-site activation. Extensive bioinformatics analysis of novel missense variants, and of the entire HGD monomer, confirmed mCSM as an effective computational tool for evaluating possible enzyme inactivation mechanisms. For the first time for AKU, a genotypephenotype correlation study was performed for the three most frequent HGD variants identified in the Suitability Of Nitisinone in Alkaptonuria 2 (SONIA2) study. We found a small but statistically significant difference in urinary homogentisic acid (HGA) excretion, corrected for dietary protein intake, between variants leading to 1% or >30% residual HGD activity. There was, interestingly, no difference in serum levels or absolute urinary excretion of HGA, or clinical symptoms, indicating that protein intake is more important than differences in HGD variants for the amounts of HGA that accumulate in the body of AKU patients.

Low-coverage massively parallel genome sequencing for non-invasive prenatal testing (NIPT) of common aneuploidies is one of the most rapidly adopted and relatively low-cost DNA tests. Since aggregation of reads from a large number of samples allows overcoming the problems of extremely low coverage of individual samples, we describe the possible re-use of the data generated during NIPT testing for genome scale population specific frequency determination of small DNA variants, requiring no additional costs except of those for the NIPT test itself. We applied our method to a data set comprising of 1,548 original NIPT test results and evaluated the findings on different levels, from in silico population frequency comparisons up to wet lab validation analyses using a gold-standard method. The revealed high reliability of variant calling and allelic frequency determinations suggest that these NIPT data could serve as valuable alternatives to large scale population studies even for smaller countries around the world. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nature methods frequencies of variants in our sample set and six different ExAC populations. Results are shown separately for (a) SNVs based on 68,326 variants; and (b) indels based on 2,909. In both cases, only those variants were used, which were simultaneously identified in each data subset with MAF higher than 5%. AFR = African/African American, AMR = American (Latino), EAS = East Asian, FIN = Finnish, NFE = Non-Finnish European, SAS = South Asian, SVK = Slovak. 2 7 SupplementaryTable 1 (uploaded as a separate Excel sheet of the Supplementary Table file):List of variants identified in the region of the CLCN1 (chloride voltage-gated channel 1, OMIM * 118425) gene in ExAC populations, dbSNP and in our data set. Only 17 of them had ExAC frequencies above 5% (highlighted in yellow). All but five were identified in our data set too with very similar calculated population frequencies. The exceptions, rs34904831, rs191902231, rs182668076, rs2280663 and rs73726622, were found to have ExAC frequencies +/-5% (depending on population). Originally three of these variants were identified in our data set too, although they were filtered out due slightly lower than 5% frequency (Suppl.Tab.1), further suggesting the feasibility of lowering our frequency restrictions. Our data contained also 89 variants missing from ExAC (highlighted in red), but found to have each of them deep intronic positions falling outside ExAC´s BED file. AFR = African/African American, AMR = American, EAS = East Asian, FIN = Finnish, NFE = Non-Finnish European, SAS = South Asian, SVK = Slovak. Supplementary Table 2 (uploaded as a separate Excel sheet of the Supplementary Table file): Verification of 87 positions in five polymorphic genomic loci of 58 randomly selected samples of our sample set from which genomic DNA was available to validation purposes. Reads from positions covered by multiple reads in individual samples (such in case of sample ID4730, where both alleles we...

We declare potential competing financial interest in the form of employee contracts (see affiliations for each author) with Geneton Ltd. that participated in the development of a commercial NIPT test in Slovakia. On the other hand, Geneton Ltd. is not a provider of this commercial test, but still continues to do basic and applied research in the field of NIPT.Minarik G and Sekelska M are employees of Medirex Inc./TrisomyTest Ltd. (the commercial providers of NIPT testing in Slovakia), their participation in the study was, however, limited to the routine NIPT testing that generated the genomic results reused in our study. The other authors declare no possible competing interests.

Supplementary materials are available at Bioinformatics online.

Noninvasive prenatal testing (NIPT) is one of the most common prenatal screening tests used worldwide. Trisomy Test® belongs to NIPT tests based on low-coverage whole-genome sequencing. In our prospective study, 7279 samples of pregnant women collected during approximately two years were analyzed. In this cohort, 117 positive cases for trisomies 21, 18, and 13 were reported. An in-house designed bioinformatic pipeline and proprietary biostatistical approach was used for the detection of trisomies. The pooled sensitivity and specificity of our test reached 99.12% and 99.94%, respectively. The proportion of repeatedly uninformative results after repeated blood draws was 1.11%. Based on the presented results, we can confirm that the Trisomy Test® is fully comparable with other commercial NIPT tests available worldwide.