Ilaria Carboni

Inner ear abnormalities in four patients with dRTA and SNHL: clinical and genetic heterogeneity

A significant number of patients affected by autosomal recessive primary distal renal tubular acidosis (dRTA) manifest sensorineural hearing loss (SNHL). Mutations in ATP6V1B1 are associated with early onset SNHL, whereas ATP6V0A4 mutations have been described in dRTA and late-onset SNHL. Enlarged vestibular aqueduct (EVA) was described in patients with recessive dRTA and SNHL, and recently, this abnormality has been associated with mutations in the ATP6V1B1 gene. In our study, we evaluated the presence of inner-ear abnormalities in four patients affected by dRTA and SNHL, characterized by molecular analysis. Two patients affected by severe dRTA with early onset SNHL showed the same mutation in the ATP6V1B1 gene and bilateral EVA with a different degree of severity. The other two presented similar clinical manifestations of dRTA and different mutations in the ATP6V0A4 gene: one patient, showing EVA, developed an early SNHL, whereas in the other one, the SNHL appeared in the second decade of life and the vestibular aqueduct was normal. Our study confirms the association of EVA and mutations in the ATP6V1B1 gene and demonstrates that mutations in the ATP6V0A4 gene can also be associated with EVA probably only when the SNHL has an early onset. The pathophysiology of SNHL and EVA are still to be defined.

Medullary sponge kidney associated with primary distal renal tubular acidosis and mutations of the H+-ATPase genes

BACKGROUND Medullary sponge kidney (MSK) is a rare congenital disease characterized by diffuse ectasia or dilation of precalyceal collecting tubules. Although its pathogenesis is unknown, the association with various congenital diseases suggests that it could be a developmental disorder. In addition to the typical clinical features of nephrocalcinosis and urolithiasis, patients with MSK show tubular function defects of acidification and concentration. These are considered to be secondary to morphological changes of collecting tubules. Primary distal renal tubular acidosis (dRTA) is a rare genetic disease caused by mutations in different genes involved in the secretion of H(+) ions in the intercalated cells of the collecting duct required for final excretion of fixed acids. Both autosomal dominant and autosomal recessive forms have been described, the latter is also associated with sensorineural hearing loss. METHODS AND RESULTS We report two patients presenting with dRTA, late sensorineural hearing loss and MSK, in whom molecular investigations demonstrated the presence of mutations of the H(+) proton pump ATP6V1B1 and ATP6V0A4 genes. CONCLUSIONS These observations, including a previous description of a similar case in the literature, indicate that MSK could be a consequence of the proton pump defect, thus can potentially provide new insights into the pathogenesis of MSK.

Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples: Nucleic Acids

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeqTM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub‐optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9–10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as “low coverage” (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeqTM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1–44.8% and 10.9–58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics.

Genetic STRs variation in a large population from Tuscany (Italy)

Allele frequencies for 17 STRs, together with some parameters of forensic interest, were estimated in a sample of 835 unrelated individuals born in Tuscany, an Italian region. These data were compared with Italian, Chinese, Kosovo Albanian, Romanian and Tunisian populations, strongly represented in this area. No significant differences in single loci were detected, except for Chinese in comparison with all the other populations.

Improving complex kinship analyses with additional STR loci

In a standard paternity testing, mother, child, and alleged father are analyzed with STR markers using commercially available kits. Since Italian civil legislation does not have thresholds to confirm a paternity, paternity is practically proven when likelihood ratio increases prior probability of paternity to posterior, accepted by court as sufficient. However, in some cases the number of markers included in a commercial kit may be insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. Additional genetic information can increase the values of the likelihood ratio regarding the detection of true parental relationships in a pedigree, while reducing the chances of false attributions (e.g. false paternities). In these cases the introduction of a 26Plex amplification system allows to examine 23–26 additional markers depending on the commercial kit used, thus increasing the statistical power of the kinship analysis. The PCR conditions were optimized for a multiplex amplification system and a new generation CE instrument. In order to demonstrate the utility of additional STRs markers, four complex kinship cases are presented.