2.1. Case presentation
A 21-year-old Caucasian woman with a 24-year-old Caucasian man as her fiancé went to Dr. Shah Veisizadeh Medical Genetics Center in Kermanshah for premarital genetic counseling. They had a cousin relationship. The man was the son of her cousin. After receiving family information, the couple's whole blood was collected for the WES test using the carrier screening approach. Both of them had no inherited disease or disorders and also, they had no familial background for certain diseases or disorders. The genetic counselor reported that they had normal phenotypes and no sign of speech, hereditary and ocular impairments.
WES revealed eight mutations including seven autosomal mutations and one x-linked mutation were found in the WES test of woman (Table 1). All mutations were checked in her fiancé and no similar mutation was found. To find the origin of the X-linked mutation, her father and mother were checked and it was found that her mother is the carrier of the mutation. In the final step, her two uncles were checked for the mutation and the results revealed that both of them had the mutation. Notably, the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. These variants are related to genes registered in the OMIM database and disorders related to autosomal recessive and X-dependent inheritance. Molecular confirmation through Sanger sequencing of the trench and examination of the transferability of his wife was performed to investigate the importance of the mentioned variant in the future pregnancies of this couple. Due to the autosomal recessive inheritance of diseases related to these genes and due to the neonatal carrier of her fiancé and the lack of mutual mutations, the risk in this regard will not affect the future pregnancies of this couple.
Table 1
WES results of the female case study.
Variant | Zygosity | Classification* |
TCTN2:NM_024809:exon15:c.1613-2A > G | het / AR | Pathogenic |
TARS:NM_152295:exon9:c.A955G:p.K319E | het / AR | Uncertain significance® |
SPEG:NM_005876:exon37:c.G9058A:p.E3020K | het / AR | Uncertain significance |
CPS1:NM_001875:exon29:c.C3539T:p.A1180V | het / AR | Uncertain significance |
MYO3A:NM_017433:exon20:c.T2208G:p.I736M | het / AR | Uncertain significance |
NNT: NM_182977:exon20:c.G2903A:p.R968Q | het / AR | Uncertain significance |
MED23:NM_004830:exon12:c.A1217C:p.K406T | het / AR | Uncertain significance |
CACNA1F:NM_005183:exon35:c.G4085A:p.R1362Q | het / XLR | Uncertain significance |
* According to the ACMG guidelines for the interpretation of sequence Variants. All the uncertain classified variant(s), listed above, have at least two moderate pathogenicity ACMG criteria and/or consistent with reported pathogenic classified variant(s) in Clinvar and literature if an affected child is born and reported based on familial history of the examined person. Het means heterozygote, AR means autosomal recessive, and XLR means x-linked recessive. |
2.2. In vitro investigations
DNA of all individuals were extracted from 2cc whole blood samples. Then, genomic DNA was fragmented and enriched for exome sequences using the SureSelect Human All Exon Kit version 6 and sequencing was performed at a minimal average coverage of 90x on an Illumina HiSeq 4000 platform. The initial sequencing component of this test was done by the Illumina Whole-Genome Sequencing Service in Macrogen (Seoul, Korea) and the alignment, variant calling, data filtering, and interpretation were performed.
Reads were aligned to the human reference sequence (GRCh37) using the Burrows-Wheeler Aligner (BWA), and variant calls were made using the Genomic Analysis Tool Kit (GATK). Variants were filtered to identify the most potential candidate variants. The evidence for phenotype-causality was then evaluated for each variant resulting from the filtering strategies. Only those variants with evidence for causing or contributing to disease are reported.
Each variant was evaluated based on the available information from the following: databases (including HGMD, ClinVar, LSDBs, NHLBI Exome Sequencing Project, 1000 Genomes, and dbSNP), published literature, clinical correlation and its predicted functional or splicing impact using evolutionary conservation analysis and computational tools (including AlignGVGD, MAPP, MutationTaster, PolyPhen-2, SIFT, and SNAP). With mean coverage of 146x, 98.8% of bases within exons were covered by at least 1 read, 94.4% of bases within exons were covered by at least 5 reads, and 84.8% of bases within exons were covered by at least 20 reads. For screening the mutations in patient’s father, mother, and uncles, sanger sequencing were performed and the represented in the Fig. 1.
2.3. In silico investigations
To conform the molecular assessments, the present report performed the homology modeling and superimposition evaluations for CACNA1F protein structure. Homology Modeling was done through SWISS MODEL and evaluated by Molprobity and ProSA online software (available at https://prosa.services.came.sbg.ac.at/prosa.php and https://prosa.services.came.sbg.ac.at/prosa.php ). Final models were vidualized by the PyMOL Molecular Graphics System (Version 2.0 Schrödinger, LLC) and also FATCAT (available at https://fatcat.godziklab.org/ ).