Non-invasive prenatal testing (NIPT): Combination of copy number variant and gene analyses using an "in-house" target enrichment next generation sequencing-Solution for non-centralized NIPT laboratory?

OBJECTIVE: Recent studies have integrated copy number variant (CNV) and gene analysis using target enrichment. Here, we transferred this concept to our routine genetics laboratory, which is not linked to centralized non-invasive prenatal testing (NIPT) facilities. METHOD: From a cohort of 100 pregnant women, 22 were selected for the analysis of maternal genomic DNA (gDNA) along with fetal cell-free DNA. Using targeted enrichment, 135 genes were analyzed, combined with aberrations of chromosomes 21, 18, 13, X, and Y. The data were subjected to specificity and sensitivity analyses, and correlated with the results from invasive testing methods. RESULTS: The sensitivity/specificity was determined for the CNV analysis of chromosomes: 21 (80%/75%), 18 (-/82%), 13 (100%/67%), and Y (100%/100%). The gene detection was valid for maternal gDNA. However, for cell-free fetal DNA, it was not possible to determine the boundary between an artifact and a real sequence variant. CONCLUSION: The target enrichment method combining CNV and gene detection seems feasible in a regular laboratory. However, this method can only be responsibly optimized with a sufficient number of controls and further validation on a strong bioinformatic background. The present results showed that NIPT should be performed in specialized centers, and that its introduction to isolated laboratories may not provide valid data.

A founder COL4A4 pathogenic variant resulting in autosomal recessive Alport syndrome accounts for most genetic kidney failure in Romani people.

Introduction: Romani people have a high prevalence of kidney failure. This study examined a Romani cohort for pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes that are affected in Alport syndrome (AS), a common cause of genetic kidney disease, characterized by hematuria, proteinuria, end-stage kidney failure, hearing loss, and eye anomalies. Materials and methods: The study included 57 Romani from different families with clinical features that suggested AS who underwent next-generation sequencing (NGS) of the COL4A3, COL4A4, and COL4A5 genes, and 83 family members. Results: In total, 27 Romani (19%) had autosomal recessive AS caused by a homozygous pathogenic c.1598G>A, p.Gly533Asp variant in COL4A4 (n = 20) or a homozygous c.415G>C, p.Gly139Arg variant in COL4A3 (n = 7). For p.Gly533Asp, 12 (80%) had macroscopic hematuria, 12 (63%) developed end-stage kidney failure at a median age of 22 years, and 13 (67%) had hearing loss. For p.Gly139Arg, none had macroscopic hematuria (p = 0.023), three (50%) had end-stage kidney failure by a median age of 42 years (p = 0.653), and five (83%) had hearing loss (p = 0.367). The p.Gly533Asp variant was associated with a more severe phenotype than p.Gly139Arg, with an earlier age at end-stage kidney failure and more macroscopic hematuria. Microscopic hematuria was very common in heterozygotes with both p.Gly533Asp (91%) and p.Gly139Arg (92%). Conclusion: These two founder variants contribute to the high prevalence of kidney failure in Czech Romani. The estimated population frequency of autosomal recessive AS from these variants and consanguinity by descent is at least 1:11,000 in Czech Romani. This corresponds to a population frequency of autosomal dominant AS from these two variants alone of 1%. Romani with persistent hematuria should be offered genetic testing.

GRN mutation in a patient with a behavioral variant of frontotemporal lobar degeneration (bvFTD).

The clinical spectrum of frontotemporal lobar degeneration (FTLD) is characterized by personality changes, language impairment, and executive function deficits. About 40% of FTLD cases have a family history of the disease, and the GRN gene is currently the most frequent genetic determinant. In cases of inherited FTLD with GRN mutations, parkinsonism is often an early sign due to greater grey matter atrophy in the caudate nucleus and bilateral atrophy in the thalamus. We investigated a female patient with signs of frontotemporal lobe atrophy and unilateral caudate nucleus atrophy on MRI. DNA was isolated from peripheral blood leukocytes and tested for GRN gene mutations. A pathogenic splice donor site mutation, c.708+1G>A, was found in the GRN gene. MRI showed unilateral caudate nucleus atrophy. This report extends the evidence for phenotypic and neuropathological heterogeneity in FTLD spectrum disorders due to splicing mutations in the GRN gene. .