In the end, millions of variants can be filtered down to a handful occurring in a small number of genes. Once the filtering reduces the number of genes and alleles to a manageable number, they can be assessed for other characteristics. First, do any of the genes have a known function or tissue expression pattern that would be expected if it were the potential disease gene? Is the gene involved in other disease phenotypes, or does it have a role in pathways with other genes in which mutations can cause similar or different phenotypes? Finally, is this same gene mutated in other patients with the disease? Finding mutations in one of these genes in other patients would then confirm this was the responsible gene in the original trio.
In some cases, one gene from the list in step 4 may rise to the top as a candidate because its involvement makes biological or genetic sense or it is known to be mutated in other affected individuals. In other cases, however, the gene responsible may turn out to be entirely unanticipated on biological grounds or may not be mutated in other affected individuals because of locus heterogeneity (i.e., mutations in other as yet undiscovered genes can cause a similar disease).
Such variant assessments require extensive use of public genomic databases and software tools. These include the human genome reference sequence, databases of allele frequencies, software that assesses how deleterious an amino acid substitution might be to gene function, collections of known disease-causing mutations, and databases of functional networks and biological pathways. The enormous expansion of this information over the past few years has played a crucial role in facilitating gene discovery of rare mendelian disorders.
Example: Identification of the Gene Mutated in Postaxial Acrofacial Dysostosis
The WGS approach just outlined was used in the study of a family in which two siblings affected with a rare congenital malformation known as postaxial acrofacial dysostosis (POAD) were born to two unaffected, unrelated parents. Patients with this disorder have small jaws, missing or poorly developed digits on the ulnar sides of their hands, underdevelopment of the ulna, cleft lip, and clefts (colobomas) of the eyelids. The disorder was thought to be autosomal recessive because the parents of an affected child in some other families are consanguineous, and there are a few families, like the one here, with multiple affected siblings born to unaffected parents—both findings that are hallmarks of recessive inheritance (see Chapter 7). This small family alone was clearly inadequate for linkage analysis. Instead, all four members of the family had their entire genomes sequenced and analyzed.
From an initial list of more than 4 million variants and assuming autosomal recessive inheritance of the disorder in both affected children, a filtering scheme similar to that described earlier yielded only four possible genes. One of these, DHODH, was also shown to be mutated in two other unrelated patients with POAD, thereby confirming this gene was responsible for the disorder in these families. DHODH encodes dihydroorotate dehydrogenase, a mitochondrial enzyme involved in pyrimidine biosynthesis, and was not suspected on biological grounds to be the gene responsible for this malformation syndrome.
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