Access to samples is one of the main challenges of studying rare inherited conditions, particularly those whose molecular basis is unknown. Every individual harbors 3-5 million genetic variants compared to the human reference sequence. Even if we winnow these down to only rare variants likely to disrupt a protein-coding gene, hundreds still remain.
The Power of Multiple Patients
One of the most efficient ways to isolate a disease-causing variant is to search for genes that are disrupted in multiple patients. Better yet, genes disrupted in multiple unrelated patients. This is nicely illustrated by Miller syndrome, one of the first disorders whose gene was identified by exome sequencing in 2010. Here’s a table I’ve adapted from that paper showing the number of candidate genes at each stage of the analysis:
The first family kindred had two affected siblings, each harboring several thousand nonsynonymous (NS), splice site (SS), or indel (I) variants in coding regions. Filtering against population allele frequency databases and looking at both siblings simultaneously winnowed this down to 237 candidate genes, which is a lot. However, incorporating data from unrelated kindreds 2 and 3 rapidly narrowed it down to less than ten genes, of which one was DHODH, the ultimate cause of disease.
Clearly, searching for recurrently altered genes in multiple patients/kindreds with a rare disorder provides enormous discriminatory power. The challenge is finding two or more willing participants with a rare condition.
The Challenge of Rare Disorders
A rare disorder that occurs in 1 out of every 100,000 individuals will affect about 40 patients per year in the United States. Most of those will go through a battery of diagnostic testing once they come to medical attention. Only a fraction will be referred to a research study, and an even smaller fraction will be enrolled. That’s effectively a handful of new cases per year. These will likely be spread out across dozens of research institutions.
Given the wide spectrum of rare conditions, it’s extremely unlikely that two patients with the same disorder would be identified (and recognized as such) at a single institution. Programs like the NIH Undiagnosed Disease Network aim to centralize the recruitment, sequencing, and analysis of rare disease cases. However, the vast majority of undiagnosed disorders are being studies by research institutions outside of such programs.
Efforts such as MyGene2 and the MatchMaker Exchange aim to facilitate data sharing among families with rare disorders and the investigators who study them. However, a number of rare disorders are still solved by good old-fashioned academic collaboration.
The RLIM/RNF12 Gene in X-linked Intellectual Disability
Case in point: in late 2016, our rare disease research program enrolled a patient with small size, hypotonia, and developmental delay — three of the most common phenotypes of patients referred to genetics. This patient, however, also had hypospadias and ambiguous genitalia. There was also a family history of congenital diaphragmatic hernia (a birth defect involving the diaphragm), which initially did not seem very important.
We performed whole-genome sequencing on the patient and his affected parents, which yielded about 6.5 million genetic variants. After running these through our comprehensive annotation/analysis pipeline, I narrowed the list to 12 recessive genes and three X-linked genes. The most interesting candidate was a hemizygous missense variant in X-linked gene RLIM, also known as RNF12. In a recent paper in the journal Molecular Psychiatry, a European-led team had reported a similar variant (p.P587R) in the same domain in two families with non-specific mild to profound ID, behavior problems, ID, microcephaly, micrognathia and cryptorchidism.
Our clinicians thought it was an interesting fit, but noted that our patient’s phenotype seemed more severe than the cases described in the paper. We discussed testing additional family members and possibly doing X-chromosome inactivation studies (XCI) on the patient’s mother. At the same time, one of our genetics clinicians reached out to the group in Germany who’d published the RLIM paper. We were hoping, at the very least, to get their impression of whether our patient resembled the RLIM cases in their cohort.
The response was unexpected but exciting: the German team was working on a follow-up paper with clinical details and functional studies on 3 large families with affected males in several generations.
They’d already performed functional studies in zebrafish and examined XCI in heterozygous females. The phenotypes were quite similar to those of our family, including (in some cases) congenital diaphragmatic hernia. The authors graciously invited us to be part of the manuscript.
The resulting paper, Pathogenic variants in E3 ubiquitin ligase RLIM/RNF12 lead to a syndromic X-linked intellectual disability and behavior disorder, is now online at Molecular Psychiatry. Its publication not only provides definitive diagnosis for our patient, but expands the phenotypic spectrum of RLIM mutation carriers and establishes the gene’s role in normal development, cognition, and behavior.
On a broader scale, this demonstrates the power of open communication and data sharing in the rare disease research community.