Exome Sequencing

Exome sequencing, also known as whole exome sequencing (WES), is a genomic technique for sequencing the entire protein-coding region of genes in a genome (known as the exome). It consists of two steps: the first step is to select only the subset of DNA that encodes proteins. These regions are known as exons  humans have about 180,000 exons, constituting about 1% of the human genome, or approximately 30 million base pairs. The second step is to sequence the exonic DNA using any high-throughput DNA sequencing technology(Ng et al.

, 2009).

One sporadic clinically suspected Goldenhar syndrome case (P10) with the full blown phenotype was selected among the OAVS cohort and Genomic DNA from the patient and his healthy parents were processed for WES trio study.

Hundreds of variants were detected in our studied family, and in depth bioinformatics analysis using variable tools and databases was performed to filter the data and categorize the variants of interest.

One missense de novo heterozygote probably pathogenic variant was identified in DGKD gene located on chromosome 2 (NM_003648.

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2:c1748 C>T, T583I / NM_152879:c.1880 C>T, T627I).This variant was not reported before in data bases and it is annotated probably damaging in different tools through our data analysis processes.

Diacylglycerol kinase delta is an enzyme that in humans is encoded by the DGKD gene. This gene encodes a cytoplasmic enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Diacylglycerol and phosphatidic acid are two lipids that act as second messengers in signaling cascades. Their cellular concentrations are regulated by the encoded protein, and so it is thought to play an important role in cellular signal transduction(Murakami, Sakane, Imai, Houkin, & Kanoh, 2003; Sakane, Imai, Kai, Wada, & Kanoh, 1996) .

Sakane et al.(Sakane et al., 1996) used degenerate PCR to isolate a novel DGK fragment, which they termed DGK-delta, from human testis mRNA. They then cloned full-length cDNAs from human hepatoma and testis cDNA libraries. Sequence analysis revealed that this gene encodes a 1,169-amino acid protein containing, in addition to sequences homologous to other DGKs, a pleckstrin homology (PH) domain and a C-terminal tail similar to those of the EPH family of receptor tyrosine kinases. Sakane et al. (1996) suggested that, because of its different structural features, this novel DGK belongs to a subfamily of DGKs distinct from the alpha, beta, and gamma DGK isoforms. Northern blot analysis showed that this gene encodes a 6.3-kb transcript most abundant in skeletal muscle but undetectable in brain, thymus, and retina. Sakane et al. (1996) expressed this novel DGK gene in COS-7 cells and observed DGK activity which was independent of phosphatidylserine, a common activator of other DGKs.

A study by Leach et al. (Leach et al., 2007) reported the involvement of DGKD gene in central nervous system development and function, whereas Chibalin et al. Concluded that DGKD deficiency causes hyperglycemia-induced peripheral insulin resistance and metabolic inflexibility, which may contribute to mild obesity later in life (Chibalin et al., 2008).

Crotty et al. (Crotty et al., 2006) found that DGKD-null mice were smaller than their wildtype or heterozygous littermates, were born with a craniofacial anomaly in the form of open eyelids similar to Egfr (131550)-knockout mice, and developed respiratory failure and died within 24 hours after birth. Dgkd knockout increased diacylglycerol accumulation, increased threonine phosphorylation of Egfr, enhanced phosphorylation of other protein kinase C (PKC; see PRKCA 176960) substrates, and increased Pkc autophosphorylation. Hence, they concluded that DGKD regulates EGFR by modulating PKC signaling.

Although DGKD gene was not reported to be implicated in OAVS or in the RA pathway; the high expression in skeletal muscles, the eye lid anomalies in DGKD-null mice and our novel probably pathogenic variant render DGKD on the scope of OAVS specially if other variants in DGKD gene are reported in new studied families with the OAVS phenotype.

Sanger sequencing confirmation followed by functional studies are highly recommended for our patient.

Another de novo heterozygous frame shift insertion with unknown protein effect in CAMKK2 gene on chromosome 12, was harbored by P10 (NM_001270486.1:c.1611_1614 C>CTT, rs7787701848). This variant was not also reported before in databases and it is considered according to ACMG guidelines as VUS(Richards et al., 2015).

CAMKK2 (CAMKK-beta) functions early in a kinase cascade that responds to increased intracellular calcium (Hsu, Tsou, Chi, Lee, & Chen, 1998).

Northern blot analysis detected variable expression of a transcript in 23 human tissues examined, with highest expression in brain and lowest expression in lung (Hsu et al., 1998).

The product of this gene belongs to the serine/threonine-specific protein kinase family, and to the Ca++/calmodulin-dependent protein kinase subfamily. This protein plays a role in the calcium/calmodulin-dependent (CaM) kinase cascade by phosphorylating the downstream kinases CaMK1 and CaMK4. CAMKK2 regulates production of the appetite stimulating hormone neuropeptide Y and functions as an AMPK kinase in the hypothalamus(Anderson et al., 2008). It also has an important role in the development of hyperalgesia and tolerance to opioid analgesic drugs, through reduction in downstream signaling pathways and mu opioid receptor downregulation (Y. Chen et al., 2008; S?nchez-Bl?zquez, Rodr?guez-Mu?oz, Montero, de la Torre-Madrid, & Garz?n, 2008) Inhibition of CAMKK2 in mice reduces appetite and promotes weight loss(Anderson et al., 2008)

Regarding the missense variant inherited from the father in FKBP15 gene (rs142775124, NM_015258.1:c.2126 T>C, K709R) with MAF 0.00002 globally /0.00006 in Africans; and through the variant analysis and annotation, we considered it as a probably benign variant owing to its inheritance from a non manifesting father and the poor implication of the gene to craniofacial development or RA pathway. FKBP15 is involved in microtubule and microfilament dynamics and has a role in correct processing of early endosomes (Viklund et al., 2009). Western blot analysis of embryonic day 18.5 mouse tissues detected Fkbp133 at an apparent molecular mass of 180 KD in brain, heart, lung, kidney, and thymus, but not in liver. Database analysis revealed orthologs of Fkbp133 in vertebrates, but not invertebrates (Nakajima et al., 2006).

A homozygous deletion in a splice region of TSPAN8 gene located on chromosome 12, was detected in the patient (rs143825158, NM_004616.2:c.577-7_577-6 TAA>T/T). This variant of unknown significance (VUS) was also found in both parents in a heterozygous pattern.

Tetraspanin-8 is a protein that in humans is encoded by the TSPAN8 gene(Szala et al., 1990).Szala et al. isolated a cDNA from an SW948 colorectal carcinoma expression library that was transfected into COS cells. They selected transfected, antigen expressing COS cells by 'panning' with the CO-029 antibody. The cDNA encoded a 237-amino acid protein with 4 putative transmembrane domains and similarity to other transmembrane proteins, such as melanoma-associated antigen ME491 (CD63; 155740), sarcoma antigen SAS (181035), and the leukocyte cell surface antigen CD37 (151523). Northern blots showed that the antigen recognized by antibody CO-029 was expressed as a 1.15-kb RNA in SW948 colorectal carcinoma cells.

Wellcome Trust Case Control Consortium (2010) confirmed a CNV in TSPAN8 gene associated with type 2 diabetes, through association testing and follow-up replication analyses(Craddock et al., 2010).

In conclusion, there is no evidence of any involvement of CAMMK2 or TSPAN8 genes in craniofacial morphogenesis or implication in RA pathway; hence their candidacy to OAVS may be week.