ESPN 54th Annual Meeting

ESPN 2022


 
The importance of reanalysis of NGS data and further functional analysis on the example of 5 patients with a clinically diagnosed hypophosphatemic ricket.
MARGARITA SHAROVA 1 SVETLANA PAPIZH 2 OLGA LEVCHENKO 1 ALEXANDRA FILATOVA 1 ANDREY MARAKHONOV 1 ANATOLIY TULPAKOV 1 MIKHAIL SKOBLOV 1

1- RESEARCH CENTRE FOR MEDICAL GENETICS, MOSCOW, RUSSIA
2- VELTISHEV RESEARCH & CLINICAL INSTITUTE OF PEDIATRICS, PIROGOV RUSSIAN NATIONAL RESEARCH MEDICAL UNIVERSITY, MOSCOW, RUSSIA
 
Introduction:

Pathogenic variants in SLC34A1 and SLC34A3 genes encoding Na/Pi transporters are identified in patients with Hereditary Hypophosphatemic Rickets with Hypercalciuria (HHRH; MIM#241530) and Idiopathic Infantile Hypercalcemia, type 2 (IIH2; MIM#616963).

Material and methods:

Five patients with hypophosphatemic ricket were referred for molecular genetic testing. Four patients underwent WGS followed by reanalysis of genomic data; NGS panel data were reanalyzed for one. Experimental investigation of the effect of two variants on splicing was performed using minigene assay and RNA analysis.

Results:

Primary NGS data analysis didn’t reveal causative variants for patients or there was only one variant in SLC34A1 and SLC34A3. Secondary investigation of NGS data revealed previously missed in-frame deletion p.91_97del in SLC34A1with global frequency 1,7% in two patients in compound heterozygous state. Intronic deletion c.925+20_926-48del101 in SLC34A3 was found in two brothers. Both variants were previously described as pathogenic. Missed synonymous VUS c.1449G>A in SLC34A1 was detected in one. Minigene assay showed that the variant leads to truncation of exon 13 by 34 nucleotides with PTC formation that allows us to reclassify the variant as likely pathogenic. Another synonymous c.846G>A variant in SLC34A3 was also missed during primary investigation. Functional study was performed by RT-PCR and identified the variant as splicing one, leading to skipping of exon 8 without frameshift. 

Conclusions:

A molecular genetic diagnosis of HHRH and IIH2 is extremely significant for early diagnosis, correct treatment optionsand genetic counseling. Reanalysis of genomic data is important not only over time, but also by different clinical bioinformaticians. Some pathogenic variants with high global frequency could be filtered out during early stages of NGS analysis. Functional analysis allows to reclassify VUS and investigate the pathogenicity previously described variants.