Among the many fields of research and medicine aided by next-generation sequencing, few have seen a greater impact than the study of rare inherited diseases. Hundreds (certainly) or thousands (probably) of new disease-gene relationships have been reported in the past several years, causing exponential growth in databases like HGMD and ClinVar. The growth is also evident from simply perusing the table of contents of peer-reviewed genetics journals, which typically feature at least a handful of new disease-gene reports in every issue.
In the early days of MassGenomics, I even tried to keep track of new gene discoveries enabled by exome sequencing, but that quickly became untenable. There’s no way to keep up. However, I think the occasional snapshot of recent discoveries can tell us a bit about how exome sequencing is being applied to rare disease research. I collected six such studies, three each from the journals of American and European human genetics (AJHG and EJHG).
I required that they be exome sequencing and convincingly implicate a single gene. From these, I summarized the phenotype, study design (i.e. who was sequenced), some notes on the analysis, the genetic finding(s), and the supporting evidence presented. Most of these were disorders with presumed recessive inheritance, but the first example is a fascinating exception.
Study 1: Gonadal Mosaicism in Intellectual Disability with Epilepsy
Ewans et al studied a family in which the parents (healthy) had six daughters, four of whom intellectual disability with epilepsy. That might have been a combination of recessive disease with very bad luck, or something else. The authors performed exome sequencing on two affected daughters and prioritized shared variants that were very rare (MAF<1% and never seen in dbSNP) and predicted to be damaging. Because the inheritance pattern was unclear, they considered all possible inheritance patterns.
The number of variants uncovered was not provided, but the authors They identified a heterozygous frameshift insertion in IQSEC2, an X-linked gene already associated with intellectual disability and seizures. All affected daughters had the insertion, but neither of the parents did. This appears to be a case of gonadal mosaicism, since the odds of a recurrent de novo mutation at the same place are vanishingly small. The parents declined further testing that might determine whose germline had the mutation (I don’t blame them).
Study 2: A Founder Mutation In Distal Myopathy And You Won’t Believe the Gene
Peric et al describe a study that was part of the MYO-SEQ project, in which exome sequencing was applied to 91 unrelated Serbian patients with unexplained limb-girdle muscle weakness and elevated serum CK levels. Of those, 19 had a predominantly distal phenotype, and 14 of them carried a specific nonsense variant believed to be a founder mutation. The gene is titin (TTN). Now you probably understand the click-baity section header.
At over 30,000 amino acids, titin is the largest known protein-coding gene in humans, and as a result, comes up in genetic analyses all the time. In cancer genomics studies, we often see it when searching for recurrently mutated genes. However, it’s considered an effect of the gene’s massive size, and usually written off. The gene is so large that trying to view it in the gnomAD database gave me a network error.
The 14 patients that carried the nonsense variant (p.Gln35879*) that, I kid you not, is in exon #362. This truncates the very end of the longest TTN transcript, so I’d be dubious of its relevance except for two facts:
- TTN is already implicated in cardiomyoapthy, with dozens of pathogenic mutations reported in HGMD, and
- All 14 Serbian patients were either homozygous for this variant, or compound-heterozygous for it and another truncating variant.
The variant appears to segregate on a ~1 megabase haplotype in individuals of Serbian ancestry, suggesting that it’s a founder mutation (i.e. can be traced back to a common ancestor).
Study 3: Siblings Discordant for Mitochondrial Myopathy
I can’t even count the number of times I’ve been in a meeting where an under-powered genetic study is being discussed, and someone says, “Maybe we’ll get lucky.” However, I *can* count the number of times we actually did. Zero. But it does happen, as illustrated by Nazli et al. They sequenced an individual with a complex encephalomyopathy / suspected mitochondrial disease and her healthy sibling.
Prioritizing variants with MAF<3% that were homozygous in the proband but not the control yielded 13 candidate variants (I have no idea why this arbitrary MAF threshold was used, or why compound hets weren’t considered). Only one variant affected a gene, TMEM65, that localizes to mitochondria. The variant itself disrupted a splice site and was heterozygous in both parents. So far, so good. To their credit, the authors carried out a series of functional assays to demonstrate the gene’s key role in mitochondrial respiratory chain function.
Study 4: Another TMEM Gene in A Rare Syndrome
Ta-Shma et al have a study that takes advantage of clever ascertainment: two unrelated families in which patients suffered a remarkably similar severe phenotype (CNS, cardiac, renal, and digital anomalies) and were the result of a consanguineous union. In family 1, an Ashkenazi Jewish pedigree, the child had unfortunately passed away, so the authors sequenced the parents to search for shared rare heterozygous variants. In family 2, an Arabic pedigree, only the mother and affected child were available.
It turns out that the unrelated patients from both families had another thing in common: homozygous truncating mutations in TMEM260. An extensive (and impressive) set of experimental follow-up work –including CRISPR/Cas9 zebrafish experiments and immunocytochemical/biochemical studies — demonstrated that the disorder arose from nonsense-mediated decay of a specific isoform of the gene.
Study 5: Consanguineous Cohort Uncovers New Gene for ID/Microcephaly
Tawamie et al had access to an entire cohort of consanguineous families affected by presumably-autosomal-recessive intellectual disability (ID). A data sharing consortium allowed them to identify homozygous missense variants that segregate with ID in two unrelated families. One was a family of Kurdish descent in which only the proband was sequenced, and regions of homozygosity were used to prioritize candidate variants. The other was a family of southern Italian origin with two affected siblings. Both were sequenced, and shared the same homozygous variant.
Given the genetic evidence that TAF13 played a role in ID, the authors performed an interesting experiment to assess its role during development. They used siRNA to suppress the gene in human neuroblastoma cell lines, and then applied RNA sequencing. Over 1,200 genes were deregulated in the knock-down cell lines compared to controls, suggesting an important role for TAF13 in development.
Study 6: Variable Phenotypes from Similar Genetic Lesions
Mingchu Xu et al conducted a very nice study of 10 affected individuals from 7 families of various ancestries. All had a similar phenotype of retinal degeneration with some additional developmental anomalies. Although unrelated, all of the affected individuals had recessive-acting truncating mutations in CWC27, a component of the spliceosome.
Two allelic mouse models (created with CRISPR/Cas9) showed that biallelic loss caused lethality and/or a severe phenotype, whereas partial inactivation caused only a retinal degeneration phenotype. This mimics the range of clinical presentations remarkably well, and highlights the complexity of the spliceosome network in human disease.
Rare Disease Genetics in 2017:
Here’s a brief table summarizing the studies discussed above:
|Phenotype||Study Design||Analysis||Gene||VarType||Inheritance||Supporting Evidence|
|Intellectual disability and epilepsy||Affected sib pair from a family with 4 probands||Shared variants, MAF<1%, present in dbSNP, impact||IQSEC2||Truncating||X-linked dominant||Segregation testing, which showed gonadal mosaicism|
|Adult onset distal myopathy||Unrelated patients of Serbian ancestry||Relevant gene list, MAF<1%, predicted impact||TTN||Truncating||Recessive||Haplotype analysis|
|Complex encephalomyopathy (mitochondrial)||Discordant siblings||Segregation, MAF<3%, Control Exomes||TMEM65||Splice Site||Recessive||Immunofluorescence/immunoblotting, siRNA knockdown, subcellular fractionation, MT function (enzymology, protein abundance, O2 consumption).|
|CNS, cardiac, renal, and digit abnormalities||2 consang. kindreds; Mom-Dad and Mom-Proband||Shared hets, q>30, depth>10x, MAF<1%, in-house controls / Homozygosity mapping, IGV, MAF<1%, Q>30||TMEM260||Truncating||Recessive||RT-PCR, CRISPR/Cas9 in vivo testing (zebrafish), Immunocytochemical and biochemical studies|
|Intellectual disability and microcephaly||Homozygosity mapping + proband; affected sib pair||Segregation, MAF<0.5%, recurrence||TAF13||Missense||Recessive||RNA sequencing after knockdown, cell proliferation/differentiation assays|
|Retinal degeneration with/without developmental anomalies||10 affecteds from 7 unrelated families of diverse ancestries||Segregation, MAF<0.5%, recurrence||CWC27||Truncating||Recessive||CRISPR/Cas9 disease modeling (mouse)|
I’m struck by the diversity of study designs — from individuals to small families to cohorts of unrelated patients — that enabled these discoveries. I’m also impressed at the careful experimental work (especially in the last four studies) that was done to functionally validate the findings. Studies like these set a high bar for publication-worthy manuscripts, but also provide useful models for unraveling the genetic basis of severe inherited conditions.