Exome Sequencing Revealed Mutations in ADAT3 and HERC2 Genes in two Sudanese Families with Syndromic Mental Retardation


 BackgroundAutosomal recessive intellectual disabilities, syndromic and non-syndromic, are of specific importance in consanguineous communities. High throughput sequencing technologies have enhanced diagnosing the Mendelian forms of intellectual disability. Mental retardation 36 and 38 are emerging clinical entities with variable presentations that extend beyond adaptive and intellectual functioning. MethodsWe used exome sequencing, bioinformatic tools and Sanger sequencing to diagnose two Sudanese families with syndromic intellectual disability. ResultsWe identified the variant NM_138422.3 (ADAT3):c.430G>A (rs730882213) in a homozygous state in two female siblings manifesting a clinical phenotype consistent with mental retardation 36. This variant was previously identified as pathogenic founder variant in multiple families from the Middle East manifesting mental retardation 36. Our patients had bullet-shaped distal phalanges, expanding the previously reported presentation. In a second family, the proband had a clinical phenotype consistent with mental retardation 38. Genetic analysis revealed the novel homozygous variant NM_004667.4 (HERC2):c.10855C>T as the potential culprit. In addition to what has been reported previously, the proband had cleft lip and palate, bulbous nose, spastic lower limbs and stones in his gallbladder.Conclusion Herein, we corroborated and extended the previously reported phenotypic spectrums associated with autosomal recessive mental retardation 36 and 38.


Abstract Background
Autosomal recessive intellectual disabilities, syndromic and non-syndromic, are of speci c importance in consanguineous communities. High throughput sequencing technologies have enhanced diagnosing the Mendelian forms of intellectual disability. Mental retardation 36 and 38 are emerging clinical entities with variable presentations that extend beyond adaptive and intellectual functioning.

Methods
We used exome sequencing, bioinformatic tools and Sanger sequencing to diagnose two Sudanese families with syndromic intellectual disability.

Results
We identi ed the variant NM_138422.3 (ADAT3):c.430G>A (rs730882213) in a homozygous state in two female siblings manifesting a clinical phenotype consistent with mental retardation 36. This variant was previously identi ed as pathogenic founder variant in multiple families from the Middle East manifesting mental retardation 36. Our patients had bullet-shaped distal phalanges, expanding the previously reported presentation. In a second family, the proband had a clinical phenotype consistent with mental retardation 38. Genetic analysis revealed the novel homozygous variant NM_004667.4 (HERC2):c.10855C>T as the potential culprit. In addition to what has been reported previously, the proband had cleft lip and palate, bulbous nose, spastic lower limbs and stones in his gallbladder.

Conclusion
Herein, we corroborated and extended the previously reported phenotypic spectrums associated with autosomal recessive mental retardation 36 and 38.

Background
Autosomal recessive intellectual disability (ARID), also known as autosomal recessive mental retardation (ARMRT), accounts for most of the cases with single gene intellectual disabilities in consanguineous communities (Hu et al., 2019). It is sub-divided to syndromic and non-syndromic depending on the presence or absence, respectively, of features not related to intellectual and adaptive functioning (Khan et al., 2016). More than 66 genes are implicated in the pathogenesis of ARID (Hu et al., 2019). Advances in genetic diagnosis and high throughput technologies have enabled delineating phenotypes associated with each gene (Harripaul et al., 2017).
ADAT3 encodes a subunit of tRNA-speci c adenosine deaminase enzyme, a heterodimeric enzyme composed of ADAT2 and ADAT3 subunits and catalyzes the deamination of Adenine to Inosine on multiple eukaryotic tRNAs (Rafels-Ybern, Attolini and de Pouplana, 2015). This deamination is important for the degeneracy of the genetic code (Gerber and Keller, 1999).
Autosomal recessive mental retardation 38 (MRT38, OMIM # 615516) is a syndrome caused by bi-allelic mutations in HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 2 gene (HERC2) and is characterized by developmental delay, abnormal gait, impaired social development and other psychiatric and non-psychiatric manifestations (Puffenberger et al., 2012).
HERC2 gene encodes a large multi-functional and multi-domain protein that belongs to the HERC family of proteins (García-Cano et al., 2019). HERC2 protein serves in ubiquitination of proteins directly, through its E3 ubiquitin ligase function, and indirectly, by activating other ubiquitin ligases (Bekker-Jensen et al., 2010). HERC2 protein is also implicated in DNA repair, cell growth, checkpoint control and genomic stability (Bekker-Jensen et al., 2010;Lai et al., 2019;García-Cano et al., 2020).
Proteomic analysis implicated multiple molecular pathways in HERC2 mediated neurodevelopmental phenotype including pathways involved in the morphogenesis of neurons in addition to mitochondrial and ubiquitin-proteosome pathways (Abraham et al., 2019). Functional studies suggested a role for HERC2 protein in brain synaptogenesis and motor coordination (Cubillos-Rojas et al., 2016;Valnegri et al., 2017).
We herein report two Sudanese families with MRT36 and MRT38 further extending the phenotypic spectrum associated with these conditions.

Methods
In this family based study we used exome sequencing and Sanger sequencing to reach the genetic diagnosis in two Sudanese families segregating intellectual disability syndromes. We collected two ml of saliva from the patients and healthy related controls using Oragene•DNA (OG-500 and OG-575) DNA kits (DNA Genotek Inc., Ottawa, ON, Canada). DNA was extracted following the prepIT.L2P manual protocol provided by the manufacturer. DNA quantity and quality were checked using NanoDrop spectrophotometer (Thermo Scienti c, Wilmington, DE, USA), Qubit uorometer (Promega, Madison, WI, USA) and standard agarose gel electrophoresis. DNA from the patients was sent for exome sequencing at the genotyping and sequencing core facility of the Brain Institute -ICM, Paris, France. Exons were captured on genomic DNA using the SeqCap® EZ MedExome Kit (Roche, IN, USA), followed by massive parallel sequencing on a Novaseq 6000 sequencer (Illumina, CA, USA). Except for aligning reads to the hg37 version of the human genome (NCBI) using Burrows-Wheeler Aligner software, we processed exome data up to the calling of variants using the Genome AnalysisToolkit software (GATK) following GATK4 best practice pipeline (Li and Durbin, 2009;McKenna et al., 2010).
We annotated and prioritized variants using software included in VarAFT annotation and lter tool (Desvignes et al., 2018). We ltered all variants with allele frequencies < 0.0001 in GnomAD genome database. First, we examined variants with predicted major structural effects; nonsense, stop loss, frameshift and splice site variants. After checking for loss of function variants, we examined missense variants annotated as pathogenic by Sift and Polyphen software and non-frame-shift variants. To verify that we had not missed strong candidate variants due to the conservative frequency lter that we used, we repeated the analysis using a frequency cut-off of 0.001 in GnomAD genome database.
We validated variants and checked their segregation in families using Sanger sequencing. Primers were designed on Genome Compiler platform. Sanger sequencing was done at the labs of Euro ns Genomics (Germany). Sanger sequencing data was visualized using Sequence Scanner Software v2.0 (Thermo Fisher Scienti c, USA).

Family F63
Patients F63-476, 34 years old, and F63-477, 30 years old, were female siblings born to a consanguineous Sudanese family. Both sisters presented with intellectual disability, strabismus and psychiatric manifestations in the form of mood swings in the elder sister and severe physical aggression in the younger.
The patients were outcomes of uncomplicated pregnancies and normal vaginal deliveries. The course of development in the two sisters was characterized by global developmental delay.
Patient F63-476 sat at the age of 2 years and walked at 7. At no time in her development did she achieve spoken language except for the vocalizations 'mum' and 'dad' achieved at the age of 4. Throughout her development, her cognitive abilities were markedly delayed compared to her healthy peers.
Patient F63-477 crawled at the age of 18 months and walked at the age of 5 years. Akin to her sister, she did not achieve spoken language apart from few vocals and had delayed cognitive abilities.
On clinical examination, both patients had dysmorphic features (up-slanted palpebral ssures, epicanthus, depressed nasal bridge and bullet-shaped distal phalanges in both siblings and frontal bossing peculiar to the younger).
Neurological examination of the two sisters revealed spasticity and hyperre exia in the upper and lower extremities with preserved power, muscle bulk and coordination. Hoffmann's sign was positive in both patients. Babinski's sign was present unilaterally in patient F63-476 and absent in patient F63-477. Both patients had bilateral pes equinovalgus and overriding toes. Both patients had bilateral esotropia but no ophthalmoplegia. Ocular cerebellar signs were absent, and no extrapyramidal features were observed. Due to lack of cooperation, proper sensory examination was not performed. Both patients were able to walk independently; albeit, with a spastic gait. Fundi were not examined. Brain magnetic resonance imaging (MRI) was normal in the two sisters apart from normal anatomical variants, cavum vergae and cavum septum pellucidum, in patient F63-477 (Fig. 1).
Analysis of exome data and segregation identi ed the homozygous variant NM_138422.3(ADAT3):c.430G > A (p.Val144Met, rs730882213) as responsible for the phenotype of the two sisters since labeled pathogenic in ClinVar database. It was not found in GnomAD genome database and had low allele frequency in GnomAD exome database, 0.0000204. We did not detect other convincing variants, homozygous or compound heterozygous, that could explain the patients' clinical phenotypes.

Family F79
Patient F79-568 was a 12 years old male born at term to a consanguineous Sudanese couple. He presented with developmental delay, abnormal gait, autistic features and behavioral disturbances. At gestation, the father was 50 years old and the mother was 36. There was no positive family history of a similar condition.
Patient F79-568 was the only child conceived after many years of marriage, and his mother preferred him born by cesarean section rather than normal vaginal delivery. He cried immediately after birth but he was hypotonic, had low birth weight and had cleft lip and palate; his lip was repaired surgically at the age of two years.
With regard to his development, he had global delay in achieving developmental milestones; he crawled at the age of 2 years, walked at 6, albeit unsteadily, never spoke, never controlled his sphincters and his intellect lagged behind his peers. Early in his childhood, patient F79-568 exhibited features of autism; lack of verbal and non-verbal communication skills corresponding to his age and was not interested in playing with peers. At the age of 4 years, he became aggressive and hyperactive.
On examination he had dysmorphic features: microcephaly -his head circumference was 38 cm, large low set ears, epicanthal fold, bulbous nose (wide prominent nasal bridge, convex nasal ridge, wide nasal base, low hanging columella and enlarged naris), high arched cleaved palate, absent and malformed teeth and brachydactyly. Examination of his lower limbs showed generalized spasticity, pes equinovalgus, exaggerated re exes at the patellar and hip adductor tendons and bilateral up-going plantar responses. Examination of the upper limbs was signi cant for reduced tone at the wrist joints and exaggerated re exes at the tendons of the biceps and brachioradialis muscles. Muscle bulk and power were preserved throughout his upper and lower extremities. Ocular motor and cerebellar examinations were normal, but his gait was spastic. During the examination, he repetitively rested his head on his right hand and starred at the space for a while (motor stereotype).
Brain MRI, done at the ages of 6 and 11 years, and karyotyping were normal. Abdominal ultrasound revealed three stones in the lumen of his gallbladder. According to his father, his gallbladder stones were detected for the rst time at the age of 4 months but never required surgical intervention.

Discussion
We herein present two Sudanese families with autosomal recessive mental retardation syndromes consistent with MRT36 and MRT38. Sudan is located in east Africa, a region known for a high genetic diversity among its population (Hollfelder et al., 2017). The genetic make-up of the Sudanese population is under opposing homogenizing, consanguinity rate > 60% in some regions, and diversifying powers, admixture and migration including migration to and from the Middle East (Daak et al., 2016;Hollfelder et al., 2017).
The variant rs730882213 that we identi ed in family F63 is a founder variant in the Middle East, and herein we report it for the rst time in Africa (Alazami et al., 2013;El-Hattab et al., 2016). Although we have not traced the origin of the variant in our Sudanese family, we can reasonably hypothesize that it is the same founder variant because of the history of genetic exchange between populations in Sudan and the Middle East.
The patients from family F63 presented with many of the previously reported common and less common features of MRT36. In this report, we added bullet-shaped distal phalanges to the skeletal deformities associated with MRT36.
In family F79, two predicted pathogenic variants, variants NM_004667.4 (HERC2):c.10855C > T and NM_021949.3(ATP2B3):c.2086C > T, segregated with the disease. In a previous study we reported Sudanese sibling patients each manifesting two independent inherited neurological disorders (Cauley et al., 2019). We rebutted the possibility of two independent disorders in patient F79-568 because a mutation in ATP2B3 was reported in a single study to cause congenital cerebellar ataxia and our patient did not manifest signs of cerebellar involvement (Zanni et al., 2012). Furthermore, the variant NM_021949.3 (ATP2B3):c.2086C > T (p.Arg696Cys) had an allelic variant, NM_021949.3 (ATP2B3):c.2086C > A (p.Arg696Ser), found in a hemizygote form in a normal control in GnomAD exomes database.
In this study, patient F79-568 presented with many of the previously reported features associated with MRT38. Despite he had neonatal hypotonia, in line with previous reports, at the age of examination, he had spastic lower limbs, exaggerated re exes and spastic gait. We also added cleft lip and palate, bulbous nose (wide prominent nasal bridge, convex nasal ridge, wide nasal base, low hanging columella and enlarged naris) and brachydactyly to the spectrum of dysmorphic features associated with MRT38. Furthermore, F79-568 had gallbladder stones at a very early age that could be part of the MRT38 phenotypic spectrum, although no link is yet established with the gene's function.

Conclusion
Studying two families from Sudan, we corroborated and extended the previously reported phenotypic spectrum associated with the autosomal recessive mental retardation 36 and 38. Further studies are necessary to delineate the full spectrum of MRT36 and MRT38 phenotypes. Declarations Ethics approval and consent to participate: informed written consents for participation were obtained from participants (or guardians of participants with intellectual disability). Ethical clearance was obtained from the ethical committee at the National University -Sudan (approval number NU-RECG200).

Abbreviations
Consent for publication: signed informed consents were obtained from participants for publication of personal and medical details included in this article. For participants with intellectual disabilities, consents for publication were signed by their legal guardians, an elder brother in the family F63 and the father in the family F79 .
Availability of data and materials: data (not including participants' personal information or identi ers) are available from the corresponding author upon reasonable request.
Competing interests: the authors declare that they have no competing interests.
Authors' contributions: AY, AMB, AAH, LEE, AEA and GS designed the study. AMB, AAH, AY and OMM evaluated the patients. AY, RA, SE, MAM, FA and IZME collected the samples and relevant data, and performed the experiments. AY, AMB, AAH and GS interpreted the results. AEA, LEE, and GS supervised the study. AY, FA and GS wrote the manuscript. All authors critically revised and approved the nal version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Funding: the study was nancially supported by the European Union (grant 779257 Solve-RD from the Horizon 2020 Research and Innovation Program, to GS). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.