الملخص الإنجليزي
Neurodevelopmental disorders are a diverse group of illnesses characterized by
impairments in personal, social, cognition, motor, or language skills as a result of
abnormal brain development. One example of a neurodevelopmental disorder is
intellectual disability. Genetic etiologies of ID were found to play a major role, and its
prevalence is estimated to be three times more in countries known for consanguineous
marriages. The burden of neurological genetic diseases in Oman is profound, especially
with the increased incidence of autosomal recessive diseases. As a result, understanding
the causes of intellectual disability will aid in providing specific counseling to families,
thereby assisting in prevention. In recent years, Next Generation Sequencing has
effectively accelerated ID research and provided novel therapeutic methods. Furthermore,
Whole exome sequencing has been shown to be a powerful, robust, and scalable approach
for discovering candidate genes, particularly in consanguineous populations.
This study aimed to perform WES on patients affected with ID that are mainly born to a
consanguineous family where the data collection was completed for 125 families with a
total number of 224 affected patients. The variants identified after WES data analyses
were prioritized, and Sanger sequencing was used for segregation analysis. Then, the
diagnostic yield and clinical impact of variants identified in patients with suspected
genetic diseases were established. Subsequently, functional characterization was carried
out for selected candidate genes. Functional analyses were performed at the RNA level
and protein level to confirm the pathogenic nature of the identified variant.
Herein, Whole exome sequencing data analysis of 125 Omani families with
neurodevelopmental disorders revealed a diagnostic rate of 31.2%. The consanguinity
rate of the 125 families included in this study was found to be 88%. Following the ACMG
guidelines of variant classification, pathogenic (P) or likely pathogenic (LP) variants were
detected in 39/125 families (31.2%), with a total number of affected individuals was 56.
Variants of uncertain significance were seen in 35/125 families (28%); these variants are
rare, predicted to be damaging, segregated with the phenotype, and explain the disease
manifestations.
Candidate genes with a possible association with the phenotype seen in our families were
detected in 30/125 (24%) with a total number of affected individuals of 64. For all variants
in candidate genes, family pedigrees were included up to 3 generations. The total number
of candidate genes identified was 28. These genes included: AATK, AP1G2, CAMSAP1,
CCDC9B, CDC42BPB, CNP, CNTROB, DNAH14, DNAJB4, DRG1, DTNBP1, EDRF1,
EEF1D, EXOSC4, FARSB, FBXO22, FILIP1, INPP4A, P2RX7, PRDM13, PTRHD1,
SCN10A, SCYL2, SUPV3L1, TACC2, THUMPD1, XPR1, ZFYVE28
Furthermore, 3 candidate genes with possible causative variants were selected for
functional characterization. In a family (10MS13800) with four affected individuals, a
homozygous 28-nucleotide frameshift deletion introducing a premature stop codon in the
PTRHD1 exon 1 was discovered. It was additionally confirmed that the aberrant transcript
escaped from the nonsense-mediated messenger RNA (mRNA) decay pathway. Real time PCR showed that mRNA expression of the mutant PTRHD1 is higher compared to
the wild type. Using the patient's primary cells, western blotting and isoelectric focusing
revealed a truncated but stable mutant PTRHD1 protein. The study provided further
evidence that PTRHD1 mutations are associated with autosomal-recessive childhood onset intellectual disability as well as spasticity and parkinsonism.
Another family (16MS3000) with four affected newborns was reported. They all
presented with congenital microcephaly which led to death within the first few months of
life. Initial research exome sequence analysis of the proband identified a highly damaging
NME1 homozygous variant as a possible novel cause of primary microcephaly phenotype
in humans. Results of In-vitro studies including western blot and immunoprecipitation
unproven NME1 variants as the cause of the phenotype. Regular reanalysis of the exome
for this family (16MS3000) uncovered the actual cause, which is the biallelic ASNS non coding variant. Reverse Transcription (RT-PCR) using the patient's primary cell line
confirmed that the aberrant transcript was stable and escape NMD-pathway. Conversely,
mutant cDNA sequencing showed that there is an abnormal insertion of 4 bp (GTAG)
from intron 6 into exon 7 in comparison to wild-type cDNA. Western blotting showed
that ASNS protein in the affected patient's lysate was not detected which indicated
aberrant degradation of the protein. Furthermore, the biochemical analysis showed that
Cerebrospinal fluid (CSF) asparagine level was not detectable. These results suggested
that the phenotype in the family is due to the ASNS variant.
