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Somatic mosaicism in focal epilepsy. Recent findings highlighted the role of somatic parental mosaicism in epileptic encephalopathies. However, somatic mosaicism has also emerged over the last few years as a prominent mechanism in the pathogenesis of lesional focal epilepsies, including focal cortical dysplasia (FCD) type 2 and hemimegalencephaly. Previous studies have identified the role of mosaicism of genes such as MTOR, TSC1/TSC2, and genes encoding components of the PI3K/AKT pathway in patients with epilepsy secondary to brain malformations. A recent study in Annals of Neurology has identified a new unrelated genetic cause of refractory non-lesional focal epilepsy, which leads us to wonder what role mosaicism may be playing in focal epilepsies without obvious findings on MRI.

Function of UGT. The UDP-galactose transporter is a nucleotide sugar transporter that pumps UDP-galactose into the ER and Golgi apparatus. Within the ER and Golgi, UDP-bound galactose is then used to add galactose residues to glycoproteins and glycolipids. If the UDP-galactose transporter is non-functional, glycolsylation cannot proceed completely, leading to a congenital disorder of glycosylation. The cartoon is modified from Glycoforum (http://www.glycoforum.gr.jp/science/word/glycoprotein/GPC03E.html) under a Fair Use Agreement and with the agreement of the authors.

An unusual suspect for focal epilepsies. Germline pathogenic variants in SLC35A2, which is located on the X chromosome, cause an epileptic encephalopathy in females characterized by intractable infantile spasms with hypsarrhythmia and significant developmental delays. The onset is usually in the first 3 months of life. Reported brain MRI findings include cerebral and cerebellar atrophy, thinning of the corpus callosum, and abnormalities of myelination. Some patients additionally demonstrate abnormal serum transferrin profiles, consistent with glycosylation defects. Rare affected males have been reported who are somatic mosaic for pathogenic variants in peripheral blood leukocytes. SLC35A2 encodes a uridine diphosphate-galactose transporter that transports galactose from the cytosol into Golgi vesicles and serves as a glycosyl donor for the generation of glycans. Both the clinical features of previously reported patients and the known function of SLC35A2 make it an unusual candidate to cause refractory focal epilepsy.

A broad clinical spectrum. Winawer and colleagues examined brain tissue of 56 patients who underwent surgical resection for refractory focal seizures and identified mosaic variants in SLC35A2 in 5 patients, four male and one female. The SLC35A2 variants were not detectable in blood in any of these individuals, indicating a postzygotic mutational event. Three patients had normal MRIs prior to surgery, and two had FCD visible on MRI. The three patients with normal MRIs had typical development and no features of SLC35A2-encephalopathy. The two males with visible FCD on MRI both had epileptic spasms and higher variant allele fractions, which may explain their more severe presentation. Interestingly, pathology of resected brain tissue revealed FCD type 1a in two of the apparently non-lesional cases.

Focal cortical dysplasia type 1a. Somatic mosaicism of genes in the mTOR/PI3K/AKT pathway has been previously reported as a cause of FCD type 2, which is characterized by disorganization of the tissue architecture with dysplastic, megalocytic neurons mixed with normal neurons. In contrast, FCD type 1 is defined by dyslamination and disorganization of the tissue architecture but with morphologically normal neurons. MRI is often considered non-lesional in patients with FCD type 1a because the cellular density of the cortex is unchanged. While several genes have been identified in FCD type 2, so far no genetic causes have been identified in patients with FCD type 1a.

Crossing the divide. The findings by Winawer and colleagues provide evidence that different genetic mechanisms involving the same genes may lead to very different phenotypes. One the one hand, germline pathogenic variants in SLC35A2 cause a severe early-onset epileptic encephalopathy, primarily affecting females. However, postzygotic somatic mutational events involving SLC35A2, confining the pathogenic variant primarily to the brain, can cause apparently non-lesional focal epilepsies with typical cognitive development. In both cases, dysfunction of SLC35A2 leads to epilepsies but with very different outcomes in terms of development. These findings bridge the divide between the genetic epileptic encephalopathies and non-lesional focal epilepsies and suggest that the underlying genes may be the same in some cases.

What you need to know. In a study of 56 patients who underwent surgical resection for both lesional and non-lesional refractory focal epilepsy, Winawer and colleagues identified somatic pathogenic variants in SLC35A2 in 5 individuals. The variant was not detectable in blood in any of the 5 patients, indicative of a postzygotic event. Variant fractions ranged from 4% – 53%, and two individuals with higher variant allele fractions displayed more severe phenotypes consistent with epileptic encephalopathy. Although not conclusive, these data suggest that a higher fraction of variant alleles in the brain may correlate with a more severe phenotype. Two patients had an identifiable focal cortical dysplasia prior to surgery. However in two of the three patients who had normal brain imaging, pathology post-surgery was consistent with focal cortical dysplasia type 1a. This is the first gene to be associated with focal cortical dysplasia type 1a.

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Epilepsy gene panel. Testing for genetic causes in human epilepsy is typically performed using gene panels. In contrast to our research-based exome studies in an academic setting, much of the gene panel testing is performed through commercial laboratories and much of the existing data is usually inaccessible to the scientific community. In a recent publication in Epilepsia, a large US-based diagnostic laboratory reports on some of their existing data on epilepsy gene panels by reporting the results of more than 8500 epilepsy gene panels – a cohort size that is more than five times larger than any prior exome or gene panel study in the epilepsy field. I was asked to write an editorial on this publication, and I also wanted summarize on our blog three key messages that you can take away from this study.

Distribution of genes. Distribution of pathogenic/likely pathogenic variants in > 8,500 gene panels. Four genes (SCN1A, KCNQ2, CDKL5, SCN2A) account for 50% of all pathogenic/likely pathogenic variants and SCN1A alone accounts for almost one fourth of all variants. 80% of the identified variant belong to only 13 gene, including SCN1A, KCNQ2, CDKL5, SCN2A, PCDH19, STXBP1, PRRT2, SLC2A1, MECP2, SCN8A, UBE3A, TSC2, and GABRG2.

Summary.The study by Lindy and collaborators looks at the diagnostic results in 8565 patients with epilepsy and neurodevelopmental disorders who were tested via a gene panel of 70 genes for epilepsies and related neurodevelopmental disorders. The authors find pathogenic or likely pathogenic variants in roughly 15% patients and identify SCN1A and KCNQ2  as the most common genes. Of the 70 genes tested, only 22 genes had a high yield of positive findings, while 16 genes did not show a single positive finding. Here are three key messages of this publication.

1 – The diagnostic yield of panel testing is 15%
With a rate of pathogenic/likely pathogenic variants of 15%, the study by Lindy and collaborators lands within the lower range of what has been previously reported. While the authors comment on the fact that their cohort was unselected, the size of the cohort speaks for itself and represents somewhat of a landmark that puts other studies into perspective. 15% is a solid estimate for gene panel studies and everything above this estimate is either due to selected cohorts or the use of other testing options.

2 – Major genes
The distribution of pathogenic and likely pathogenic variants across the 70 genes tested in the study by Lindy and collaborators looks like a Pareto distribution (80/20). More than 80% of all variant are explained by the top 20% genes. SCN1A alone almost accounts for one fourth of all pathogenic/likely pathogenic variants; the top four genes (SCN1AKCNQ2, CDKL5, SCN2A) account for a little more than 50% of all variants. A small 25-gene panel would have captured more than 90% of all variants, accounting for a diagnostic yield of 14% in the larger cohort. This observation argues for the use of smaller gene panels as a first-tier screening given that a good amount of the genetic diagnoses that will be found by a panel within a limited number of genes.

3 – Genes without evidence
While there are genes with a very high number of variants, the study by Lindy and collaborators also identified a large number of genes without any pathogenic or likely pathogenic variant. These genes include ATP6AP2CACNB4, CHRNA2, CSTB, CTSD, DNAJC5, EFHC1, FOLR1, GATM, GOSR2, LIAS, MAGI2, NRXN1, PRICKLE1, SLC25A22, and SRPX2. In my commentary, I provided two explanations for this observation. In fact, some of these genes may be extremely rare and may be tested in patients with a clinical diagnosis through other means. This applies to the established genes for Progressive Myoclonus Epilepsies including CSTB and GOSR2. It is also important to mention that the NGS methodology used in this study does not detect the most common pathogenic variant in CSTB, a dodecamer repeat expansion. On the other hand, some of the genes without identified pathogenic/likely pathogenic may not be disease genes at all. These genes were once thought to be good candidates, but further evidence for pathogenicity has not emerged. This applies to genes such as EFHC1, MAGI2, or SRPX2. These genes will probably be removed from diagnostic panels in the near future given the lack of evidence for a disease association. Basically, these genes are not disease genes and holding on to them in an indefinite candidate status despite lack of confirmatory evidence will eventually create more harm than good.

What you need to know. With the current study by Lindy and collaborators, the epilepsy field has taken a leap forward. Testing 70 genes in more than 8,500 patients with epilepsy provided clear evidence for common epilepsy genes versus unconfirmed candidates and indicated that up to 15% of patients can receive a genetic diagnosis based on testing of a limited number of genes. There are other aspects of this study that I did not include in this post and may discuss in the future, namely the role of deletion/duplication testing for each gene, recurrent variants, and the rate of inherited versus de novo variants.

Caveats. One important issue to mention is that some genes that are presumably not all that rare were not included in the study by Lindy and collaborators. For example, CHD2 and DEPDC5 were not among the 70 genes analyzed in their study.  A further limitation of the publication by Lindy and collaborators is the lack of detailed phenotypic data. However, the size of their study alone provided important insights into the genetic architecture of human epilepsy.

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Epilepsy gene panel. Testing for genetic causes in human epilepsy is typically performed using gene panels. In contrast to our research-based exome studies in an academic setting, much of the gene panel testing is performed through commercial laboratories and much of the existing data is usually inaccessible to the scientific community. In a recent publication in Epilepsia, a large US-based diagnostic laboratory reports on some of their existing data on epilepsy gene panels by reporting the results of more than 8500 epilepsy gene panels – a cohort size that is more than five times larger than any prior exome or gene panel study in the epilepsy field. I was asked to write an editorial on this publication, and I also wanted summarize on our blog three key messages that you can take away from this study.

Distribution of genes. Distribution of pathogenic/likely pathogenic variants in > 8,500 gene panels. Four genes (SCN1A, KCNQ2, CDKL5, SCN2A) account for 50% of all pathogenic/likely pathogenic variants and SCN1A alone accounts for almost one fourth of all variants. 80% of the identified variant belong to only 13 gene, including SCN1A, KCNQ2, CDKL5, SCN2A, PCDH19, STXBP1, PRRT2, SLC2A1, MECP2, SCN8A, UBE3A, TSC2, and GABRG2.

Summary.The study by Lindy and collaborators looks at the diagnostic results in 8565 patients with epilepsy and neurodevelopmental disorders who were tested via a gene panel of 70 genes for epilepsies and related neurodevelopmental disorders. The authors find pathogenic or likely pathogenic variants in roughly 15% patients and identify SCN1A and KCNQ2  as the most common genes. Of the 70 genes tested, only 22 genes had a high yield of positive findings, while 16 genes did not show a single positive finding. Here are three key messages of this publication.

1 – The diagnostic yield of panel testing is 15%
With a rate of pathogenic/likely pathogenic variants of 15%, the study by Lindy and collaborators lands within the lower range of what has been previously reported. While the authors comment on the fact that their cohort was unselected, the size of the cohort speaks for itself and represents somewhat of a landmark that puts other studies into perspective. 15% is a solid estimate for gene panel studies and everything above this estimate is either due to selected cohorts or the use of other testing options.

2 – Major genes
The distribution of pathogenic and likely pathogenic variants across the 70 genes tested in the study by Lindy and collaborators looks like a Pareto distribution (80/20). More than 80% of all variant are explained by the top 20% genes. SCN1A alone almost accounts for one fourth of all pathogenic/likely pathogenic variants; the top four genes (SCN1AKCNQ2, CDKL5, SCN2A) account for a little more than 50% of all variants. A small 25-gene panel would have captured more than 90% of all variants, accounting for a diagnostic yield of 14% in the larger cohort. This observation argues for the use of smaller gene panels as a first-tier screening given that a good amount of the genetic diagnoses that will be found by a panel within a limited number of genes.

3 – Genes without evidence
While there are genes with a very high number of variants, the study by Lindy and collaborators also identified a large number of genes without any pathogenic or likely pathogenic variant. These genes include ATP6AP2CACNB4, CHRNA2, CSTB, CTSD, DNAJC5, EFHC1, FOLR1, GATM, GOSR2, LIAS, MAGI2, NRXN1, PRICKLE1, SLC25A22, and SRPX2. In my commentary, I provided two explanations for this observation. In fact, some of these genes may be extremely rare and may be tested in patients with a clinical diagnosis through other means. This applies to the established genes for Progressive Myoclonus Epilepsies including CSTB and GOSR2. It is also important to mention that the NGS methodology used in this study does not detect the most common pathogenic variant in CSTB, a dodecamer repeat expansion. On the other hand, some of the genes without identified pathogenic/likely pathogenic may not be disease genes at all. These genes were once thought to be good candidates, but further evidence for pathogenicity has not emerged. This applies to genes such as EFHC1, MAGI2, or SRPX2. These genes will probably be removed from diagnostic panels in the near future given the lack of evidence for a disease association. Basically, these genes are not disease genes and holding on to them in an indefinite candidate status despite lack of confirmatory evidence will eventually create more harm than good.

What you need to know. With the current study by Lindy and collaborators, the epilepsy field has taken a leap forward. Testing 70 genes in more than 8,500 patients with epilepsy provided clear evidence for common epilepsy genes versus unconfirmed candidates and indicated that up to 15% of patients can receive a genetic diagnosis based on testing of a limited number of genes. There are other aspects of this study that I did not include in this post and may discuss in the future, namely the role of deletion/duplication testing for each gene, recurrent variants, and the rate of inherited versus de novo variants.

Caveats. One important issue to mention is that some genes that are presumably not all that rare were not included in the study by Lindy and collaborators. For example, CHD2 and DEPDC5 were not among the 70 genes analyzed in their study.  A further limitation of the publication by Lindy and collaborators is the lack of detailed phenotypic data. However, the size of their study alone provided important insights into the genetic architecture of human epilepsy.

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Genetic literacy. Sometimes important milestones don’t feel like much when you eventually reach them. Last Thursday, I woke up sleep-deprived after working on a grant all night and found an NCBI update in my mailbox. Primer Part 2 of the genetic literacy series of ILAE Genetics Commission was now published in Epilepsia and available on PubMed. Finally, both the introductory primers of the genetic literacy series are out – Part 1 dealing with the building blocks including general concepts of epilepsy genetics and epidemiology and now Part 2 about the paradigm shifts that were introduced with the advent of massive parallel sequencing. Both publications were revised and re-written over and over again to fit the overall didactic mission of the literacy series, an effort that takes approximately 10x as long as writing a typical review. But finally, as of May 10, 2018, both Primers are now in their final shape, published and open access to the international epilepsy community. And here is just a quick overview of what this paradigm shift is really about.


Figure 1. Example of interpretation process for determining the clinical relevance of genetic variants described in the vignette (recurrent variant refers to variants previously seen in individuals with a similar phenotype). ACMG, American College of Medical Genetics and Genomics; VUS, variant of uncertain significance [adapted from https://www.ncbi.nlm.nih.gov/pubmed/29741288]

A personal view. Let me quickly recapitulate why the publication of Primer Part 2 is such an important issue for me personally – I sometimes like to come back to our initial tradition on Beyond the Ion Channel to share some of my personal experiences as a clinician-researcher. During my transition from Europe to the US between 2014 and 2017, I had three relatively stubborn projects that dragged out seemingly indefinitely – three projects that kept sitting in the back of my mind where I struggled to make significant progress and after my transition to Penn faculty in July 2017, I gave myself 12 months to complete them. The first project was DNM1, the epilepsy gene that we pulled out of the noise of large exome studies and subsequently defined as a novel phenotype. Secondly, there was PMPCB, the gene for a rare mitochondrial epileptic encephalopathy where it took us several years to identify further patients and which required us to go through complex functional studies. And finally, there was Primer Part 2, our didactic publication on epilepsy genetics that you’re looking at now.

Case-oriented. Our cases in the Genetic Literacy Series start with a case vignette – for Primer Part 2, we presented a case that is representative of scenarios in our epilepsy genetics clinic and many other neurogenetics clinics around the world. You are seeing a young boy with an unexplained epileptic encephalopathy and variants in SCN9A, EFHC1, SPTAN1, KCNQ1, SCN2A, and TBC1D24 that were identified on past genetic testing a few years ago and were considered of uncertain significance. We then guide the reader through the modern concepts of assessments of gene validity, variant pathogenicity, and clinical correlation. The figure above summarizes the detective work of variant interpretation in this patient that eventually led us to the realization that the SCN2A variant seen in this patient has acquired additional evidence since it was first reported out and is likely the cause of the patient’s disease.

A typical case. Our main reason for putting together Primer Part 2 in this format is the paradigm change that we have alluded to in the title of our manuscript. While we are still struggling to obtain genetic testing in a significant number of patients, in other patients who do undergo genetic testing, a second set of problems is emerging – the incorrect interpretation of genetic testing and reliance on outdated interpretation. Basically, results of genetic testing are not static over time. Negative and uncertain test results should ideally be reviewed and re-interpreted every 12-24 months. In fact, the “reportedly negative testing in the past” is one of the more common causes of false information that we see in clinical practice.

Summary. With the Primer Part 2, we intended to provide the clinician with a guideline to understand and question results from genetic testing to enable correct interpretation. The important thing for me to emphasize is that part of the interpretation will always be with the clinician. In the same way that we review MRIs in person, we should have a basic understanding of how genetic data is analyzed. In the same way that we don’t expect the same degree of scrutiny from the overnight radiologist who reads urgent MRIs and CTs compared to the detailed radiological discussion in a surgical case conference, we should be aware that the epilepsy-specific gene and variant interpretation will always be variable and that it will be our task to remain vigilant and skeptical to make sure that we get the best information for our patients and for the treatment choices that we are making.

Where to go from here. After reading Primer Part 2, you will hopefully be familiar with some of the current concepts that are used for gene and variant interpretation – you will understand why we are not concerned about a variant in EFHC1 in our patient as the gene lacks validity and why the SPTAN1 variant initially looks like a good candidate, but is ruled out because of the frequency of this variant in the population. You will also have been confronted with the relatively formal ClinGen vocabulary that is used in the diagnostic space. If you feel that your interest is piqued on how the process of gene and variant curation is playing out on a day-by-day basis, you might be interested in learning more about the ClinGen Epilepsy Gene Curation Expert Panel that is tasked with epilepsy gene curation and providing gene-specific information in a format that is typically used in a diagnostic setting. The clinical epilepsy world and the diagnostic world are drifting more and more apart and the ClinGen Epilepsy Gene Curation Expert Panel is currently one place where we speak both languages.

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Conventional wisdom. Trio whole exome sequencing has been successful over the last five years in identifying underlying genetic etiologies in nearly 50% of patients with epileptic encephalopathies, which is largely owing to the genetic architecture of these conditions. The vast majority of these genetic epilepsies are caused by apparent de novo variants that are present in the patient but not in the mother or father. The conventional wisdom is that the recurrence risk in future pregnancies for parents of an affected child is low to non-existent and traditionally we have quoted a ~1% recurrence risk for future pregnancies. However, a new study published in the New England Journal of Medicine turns this conventional wisdom on its head, identifying detectable somatic mosaicism in approximately 10% of parents tested, which has implications for how we counsel families of children with epileptic encephalopathies – and potentially other genetic conditions due to de novo variants as well.

Mechanism of Somatic Mosaicism. Early in embryonic development of the parent, a single cell spontaneously acquires a pathogenic variant. Every cell that arises from that single cell also carries this pathogenic variant, but all other cells in the embryo do not. The presence of two distinct cell populations in the body is referred to as “somatic mosaicism”. The somatic mosaic parent is typically unaffected or mildly affected. The pathogenic variant is also present in the sperm or egg cells and can therefore be passed on to a child during pregnancy. This child has the pathogenic variant in all cells in his/her body and is affected and non-mosaic. Because the pathogenic variant is present in the sperm or egg cells of the mosaic parent, there is a chance that future pregnancies may also be affected [modified from https://commons.wikimedia.org/wiki/File:Parental_Mosaicism.svg under a CC VY 3.0 license]

Higher risk than previously thought. Myers and colleagues performed an assay called single-molecule molecular inversion probes, looking at 33 specific genes in patient-parent trios, where a child is affected with a confirmed genetic epileptic encephalopathy. Single-molecule molecular inversion probes have the advantage of being highly specific to the target of interest and, importantly, being able to detect variants occurring at very low frequencies, which traditional methods such as Sanger sequencing are unable to do. Testing DNA extracted from blood or saliva, Myers and colleagues identified mosaicism for the genetic variant in a parent from 10/120 families. Parental mosaicism was found in approximately 10% of families with children with an apparent de novo SCN1A variant, consistent with previous findings. Parental mosaicism was also found in CACNA1ADNM1GNB1, KCNT1KCNQ2SCN8A, and SLC6A1. Two additional families had inferred mosaicism due to a second affected sibling also carrying the pathogenic variant found in the proband (SYNGAP1KIAA2022) but no variant detected in the parents. Altogether, 12/120 (10%) families had evidence of parental mosaicism, which is 10x higher than the risk of mosaicism that is traditionally quoted when counseling for de novo variants. Four of the twelve mosaic parents had reported a previous history of seizures, including febrile seizures, which was milder than their affected child’s phenotype. Importantly, in 6/12 families, a second affected sibling also carried the same variant as the proband.

Mosaicism explained. When we refer to “mosaicism” in genetics, we mean that a person has two or more unique cell populations. In this case, the parent of an affected child has some cells that do not carry the pathogenic variant and a small proportion of cells that do carry the pathogenic variant. The variant in the parent likely occurred spontaneously in a single cell when the parent was an embryo, and all subsequent cells that arose from that cell also contain that genetic change. Because the mosaic parents all have affected children, the variant is presumed to be present in gonadal tissue as well, although it is not possible to test this tissue to confirm. Myers and colleagues tested DNA from peripheral blood and saliva tissue from parents and found highly variable levels of mosaicism, ranging from 1.4-30%. It’s possible that other mosaic parents were not detected because mosaicism may be present in other untested tissue types.

Counseling and practical implications.The genetic counseling implications from this study are significant. As genetic counselors and physicians who counsel families with apparent de novo variants, we need to consider how we approach these findings and what information we provide to families. The <1% risk of recurrence is likely not an appropriate recurrence risk estimate anymore, especially for families who may wish to have additional children. In cases with a mosaic parent, a precise recurrence risk calculation cannot be given, but it may approach 50%, as in autosomal dominant disorders. The practical implications are that we should consider reframing the recurrence risk estimates, inform families about the possibility of undetected parental mosaicism, and offer a referral to a prenatal genetics professional for any families who may wish to discuss prenatal or preimplantation genetic testing options for future pregnancies.

Future implications. As a genetic counselor, my ideal scenario would be for these findings to spur development of a sensitive diagnostic test that could be offered to families of children with an apparent de novo variant to help better identify mosaic parents, where this information would be useful – i.e. for families who are considering having additional children and who would use this information in making reproductive decisions. This would allow us to better refine recurrence risk estimates and help identify families who may benefit from prenatal or preimplantation genetic counseling. My concern is that currently cases of parental mosaicism may not always be reported by diagnostic laboratories even if they may be suspected, since many laboratories are not validated to return this information for parents. Parental mosaicism is likely not confined to genetic epilepsies – this phenomenon is likely seen in other sporadic genetic conditions resulting from de novo variants. Further research in other non-epilepsy fields of genetics are needed to see if these findings can be replicated in other genetic conditions.

What you need to know. In a recent study in the New England Journal of Medicine, Myers and colleagues performed analysis of parental mosaicism among 120 patient-parent trios of genetic epileptic encephalopathies and identified parental mosaicism in 10% of families. Some mosaic parents had reported a history of single seizures, and in many families a second affected sibling was born who also carried the pathogenic variant. We currently quote a recurrence risk in future pregnancies of ~1% for patients with apparent de novo variants, but these findings suggest that the real recurrence risk may be as high as 50%. Referral to a prenatal genetics professional may be appropriate for a family of a child with a confirmed genetic epilepsy, if the family is considering having additional children.

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Unravelling the BAFME mystery. The mystery surrounding Benign Adult Familial Myoclonic Epilepsy (BAFME) – also known as Familial Adult Myoclonic Epilepsy (FAME) or Familial Cortical Myoclonic Tremor and Epilepsy (FCMTE) – has persisted for years. BAFME is an autosomal dominant neurological disorder characterized by adult onset of myoclonic/cortical tremor and infrequent seizures. The clinical course is typically considered to be benign. Linkage studies have shown linkage to several regions including 8q24, 2p11.1-q12.2, 3q26.32-q28, and 5p15. A recent publication identified a variant in CTNND2 segregating with disease in a Dutch family with BAFME3, although it remains to be determined how broadly applicable CTNND2 variants are in other individuals with BAFME. Now in an elegant set of experiments by Ishiura and colleagues, a significant proportion of BAFME appears to be solved and is due to expansions of pentanucleotide intronic sequences in SAMD12.

Accumulation of RNA Foci in BAFME. A simplified schematic diagram of how RNA foci accumulate in patients with TTTTA and TTTCA repeat expansions. The RNA contains an abnormal number of repeat expansions. This RNA then takes on an abnormal secondary structure. This abnormal RNA then recruits and sequesters RNA binding proteins. These clusters form aggregates called RNA foci that accumulate within the nuclei in cortical neurons and Purkinje cells. These RNA foci have toxic effects within the cell. RNA foci only containing TTTCA repeats have been observed in autopsied brain tissue of patients with BAFME.

A surprising repeat expansion disorder. Ishiura and colleagues identified expansions of non-coding TTTCA- and TTTTA-sequences in an intron of SAMD12 in 49/51 (96%) Japanese families with BAFME1, which links to chromosomal region 8q24. The repeat sizes were highly variable, ranging from 440 to 3680 repeats. Somewhat unsurprisingly, larger expansion sizes were correlated with earlier onset of seizures and myoclonic tremor. This is similar to what we observe in other repeat expansion disorders including Huntington Disease, although curiously not Unverricht-Lundborg Disease, another repeat expansion-associated myoclonic epilepsy. Four individuals homozygous for the SAMD12 repeat expansion were observed in the cohort, and these patients experienced younger age of onset and more severe disease progression, including impairment in walking and cognitive decline, which was not observed among heterozygotes. Two families were not found to have SAMD12 intronic pentanucleotide repeat expansions. However, affected individuals from these two families were found to have TTTTA and TTTCA repeat expansions in two other genes: one family had expansions in TNRC6A and one in RAPGEF2. TTTTA expansions in all three genes were identified in a small percentage (5% or less) of healthy controls; however no control individuals were found to have TTTCA expansions in SAMD12, TNRC6A, or RAPGEF2, suggesting that TTTCA repeat expansions may play a primary role in disease etiology.

What does SAMD12 do? The answer is: it may not actually matter. Little is known about SAMD12, which encodes the Sterile Alpha Motif Domain Containing 12 protein. Western blot analysis in available brain tissue showed slightly but significantly decreased SAMD12 expression levels when compared to controls, suggesting a possible role of the protein in disease. However, the authors suggest that the disease mechanism may lie at the RNA- rather than the protein-level, particularly since the two SAMD12-negative families were also found to have TTTCA- and TTTTA-repeat expansions in other genes. Examination of autopsied brain tissue identified RNA foci, containing UUUCA sequences but not UUUUA sequences, in cortical neurons and Purkinje cells in patients but not in controls. RNA foci result when an RNA molecule contains an expanded number of repeats. RNA molecules containing an extra number of repeats are retained in the cell’s nucleus, where the RNA molecules take on an abnormal structure and isolate other RNA binding proteins along with them. These aggregates – called RNA foci – have toxic effects within the cell. RNA foci have also been observed in other neurodegenerative disorders caused by repeat expansions, such as C9ORF72 in ALS/Frontotemporal Dementia and DMPK in myotonic dystrophy. These findings suggest that BFAME is a so-called “RNAopathy”, and that RNA-mediated toxicity of RNA products containing UUUCA sequences may be the underlying pathomechanism.

Implications for other epilepsies. The findings from this study are fascinating and immediately lead us to question what we can learn from them. The non-coding pentanucleotide repeat expansions identified in patients in this study would likely not be detected by short read sequencing or whole exome sequencing, which has become the standard diagnostic and research-based sequencing methodology currently. Could intronic repeat expansions be playing a role in other unsolved presumed genetic epilepsies, including the genetic generalized epilepsies? Although we are increasingly successful in identifying underlying genetic causes in patients with severe, early-onset epilepsies, we may need to explore new methodologies – including whole genome sequencing and/or RNAseq – to crack the difficult to solve cases of the future.

What you need to know. BFAME1, linked to 8q24, is due to intronic TTTCA- and TTTTA-repeat expansions within the SAMD12 gene. Larger repeat sizes are correlated with earlier age of onset. Expression of RNA containing these repeat expansions likely lead to RNA foci in cortical neurons and Purkinje cells, suggesting that BFAME is an RNAopathy and that the underlying pathomechanism is related to RNA-mediated toxicity of RNA products containing the repeated sequences.

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MPP. Mitochondria are indispensable for cellular energy production and require constant protein import, as most mitochondrial genes are encoded in the nucleus. In order for proper targeting, mitochondrial proteins have a specific presequence, which is removed once a protein has found its way into the mitochondria. This function is accomplished by the mitochondrial processing peptidase MPP, which is encoded by the PMPCA and PMPCB genes. In a recent publication in the American Journal of Human Genetics, we identified PMPCB as a novel gene for a complex neurodegenerative condition in childhood and discovered a new disease mechanism for neurological disorders. However, epileptic encephalopathy that initially led to the inclusion of our initial RES study was only one extreme of an unusual disease spectrum. 

Figure. PMPCB dysfunction in childhood neurodegeneration and epilepsy results in reduced function of the mitochondrial preprocessing peptidase MPP. This protease cuts off the presequence that guides mitochondrial proteins to the mitochondrial matrix – MPP is the mitochondrial box cutter that opens up the packages addressed to the mitochondrial matrix.

Mitochondrial disease. In clinical practice, we typically assume that we do not encounter mitochondrial disease too often in patients with epileptic encephalopathies. We primarily assume ion channel diseases or disorders of synaptic transmission as main disease mechanisms. However, a subset of mitochondrial diseases can present with primary CNS presentation, defying the general assumption that mitochondrial diseases present with multisystem disease and clear biomarkers such as elevated lactate. Therefore, particularly in patients with regression or worsening during febrile illnesses or crises, mitochondrial diseases are always a differential diagnosis. This blog post is about the discovery of an unusual mitochondrial disorder that presented with epileptic encephalopathy in one of the initial EuroEPINOMICS families, which eventually led us to recognize a wider range of neurodegenerative conditions with prominent cerebellar atrophy.

The mechanism of PMPCB. When we first considered PMPCB as a candidate gene in a family from the EuroEPINOMICS project, we felt ambivalent about this gene as the cause of the disease. Little was known about this gene at the time, and the suspicious variant was a homozygous missense variant in a consanguineous Kurdish-Iraqi family – we typically encounter this class of variants on a regular basis and do not pay too much attention to them, as we typically see a large number of these variants in consanguineous families. On the other hand, PMPCB was a relatively intolerant gene – we had never seen variants in this gene before, which made us suspicious. When we connected with the Voegtle lab in Freiburg, Germany, it dawned on us why PMPCB variants occur so rarely – it encodes one subunit of MPP, the mitochondrial preprocessing protease, which is so critical to cellular functioning that it tolerates little variation. Basically, MPP is a mitochondrial box cutter that opens up the packages addressed to the mitochondrial matrix by removing a presequence. Yeast is usually a very robust single cell organism that tolerates a lot of genetic and environmental stress. Therefore, we were extremely surprised that the variant in the EuroEPINOMICS family basically took away the yeast’s ability to grow even under the most favorable conditions. When we examined patient cells from the affected individuals in the EuroEPINOMICS, we realized that mutated MPP is unable to fully process one of the most studied targets, the Frataxin protein that is involved in Friedreich’s ataxia. Mitochondria are no longer able to fully activate all the proteins addressed to them and unopened packages and letters keep accumulating. The mitochondrial box cutter has a very limited margin of error.

The breakthrough. The next step in the discovery of PMPCB occurred when we started collaborating with the Van Hove lab in Colorado, who had identified a patient with compound heterozygous PMPCB variants. Interestingly, this patient had a different phenotype with prominent cerebellar neurodegeneration. However, this patient did not have the epileptic encephalopathy that was initially seen in the EuroEPINOMICS family – epilepsy was in fact only the tip of the iceberg that made us realize a much wider range of phenotypes afterwards. We then soon identified two other patients with similar neurodegenerative phenotypes and variants that overlapped between patients. Biochemical studies showed mitochondrial dysfunction, including elevated lactate and impairment of the respiratory chain complexes. In addition, these variants did not lead to a lethal yeast phenotype and could finally be modelled.

The PMPCB yeast phenotype. It took me a while to understand how precisely PMPCB dysfunction can be analyzed in yeast. Yeast can be grown at low and high temperatures and in yeast strains carrying patient mutations, the PMPCB phenotype only becomes evident when yeast are stressed at higher temperatures. This situation mimics the increased energy demand with infections or with growth, possibly explaining why PMPCB-related conditions only manifest with infections or increased cellular demands during infancy. In addition, we were able to assess how MPP dysfunction affects various cellular functions. Interestingly, we found a very early and surprisingly strong impairment of iron-sulfur cluster biosynthesis. Iron-sulfur proteins are involved in a variety of cellular processes, most importantly the electron transport chain.  PMPCB dysfunction is so dramatic that it impacts the generation of these proteins almost immediately once yeast cells are stressed with higher temperatures.

MPP diseases. Identifying the role of PMPCB dysfunction almost took a decade and during this time, mutations in PMPCA have been discovered in patients with cerebellar ataxia. PMPCA encodes the non-catalytic subunit of MPP that is involved in recognizing the protein needs to be cleaved – PMPCA is the handle of the mitochondrial letter opener while PMPCB is the blade. Taken together, both disorders affecting MPP function share a common theme – prominent cerebellar involvement in the absence of multisystem disease. The latter is surprising given the known presentations of other diseases that impair iron-sulfur cluster biosynthesis. Understanding why MPP dysfunction has such a strong effect on the cerebellum is not understood, but it may provide some insight on why other brain regions are somewhat protected against MPP dysfunction.

What you need to know. Bi-allelic variants in PMPCB are a novel cause of neurodegeneration in early childhood, and some patients can have prominent epileptic encephalopathy. While the impairment of mitochondrial function is due to an impairment of iron-sulfur cluster biosynthesis, patients with PMPCB-related disorders have not been found to have systemic symptoms. Prominent cerebellar atrophy in early childhood is one of the most specific features of PMPCB-related disorders.

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Sodium channel. Voltage-gated channels for sodium ions are a crucial component of helping neurons depolarize and repolarize in a way that enables generation of action potentials. However, in order to function properly, voltage-gated ion channels co-exist in a fragile balance, and genetic alterations leading to functional changes in these channels are known causes of disease. SCN1A, SCN2A, and SCN8A have been implicated as causes for human epilepsy. However, SCN3A encoding the Nav1.3 channel, one of the most obvious candidates, could not be linked to disease so far. In a recent publication, we were able identify disease-causing mutations in this major neuronal ion channel. Interestingly, patients with an early onset and the most severe presentation had a prominent gain-of-function effect that responded to known antiepileptic medications.

Figure. Variants in SCN3A identified in our study (Zaman et al., 2018) and previous studies. Three variants found in four patients (p.I875T x2, p.P1333L, p.V1769A) were found to be de novo. A prominent gain-of-function was observed for p.Ile875Thr and p.Pro1333Leu. Both patients with the p.Ile875Thr had diffuse polymicrogyria.

Nav1.3. Voltage-gated sodium channels play a major role in tissues involved in the generation and propagation of electrical signals, most prominently the skeletal muscle, the heart muscle and, most importantly, the brain. There are nine different sodium channels in humans and four voltage-gated sodium channels expressed on neurons including the Nav1.1 (SCN1A), Nav1.2 (SCN2A), Nav1.3 (SCN3A), and Nav1.6 (SCN8A) channels. The naming of sodium channel is often a source for confusion between scientists involved in functional studies and genetic studies. Nav1.x refers to the protein, SCNxA to the gene, but for some sodium channel genes such as SCN8A, the numbering is off.  Out of this quad of brain-specific sodium channels, three channels have been associated with epilepsy and neurodevelopmental disorders so far. SCN3A had been the missing sodium channel that was suggested in some previous studies, but never arrived at the same degree of certainty as the other sodium channels including SCN1A, SCN2A, and SCN8A. Nav1.3 encoded by the SCN3A gene is highly expressed in the developing brain, but its expression drops to basically insignificant levels postnatally.

Gain-of-function. In our recent study by Zaman and collaborators, we identified four patients with de novo mutations in SCN3A, including two patients with a recurrent variant. In contrast to previous studies, which indicated a role of SCN3A in milder neurodevelopmental disorders, the four patients described in our study had severe epilepsy starting in the first year of life. Functional studies identified a prominent gain-of-function effect of these mutations, indicating that increased channel function was the molecular mechanism behind the severe presentation in patients with SCN3A epileptic encephalopathy.

Nav1.3 expression. Looking at the expression pattern of SCN3A, this mutational mechanism is somewhat evident. For a gene with a very low expression after birth, only a significant increase in channel activity can result in hyperexcitability. Basically, in patients with SCN3A encephalopathy, the overactive Nav1.3 channel adds another sodium channel-related function that is not present in the typically developing brain – and the hyperexcitable network produces the epileptic encephalopathy. A related mechanism was shown for some variants in the SCN2A and SCN8A genes, as well, even though the mechanisms can only be compared to a certain degree. In vitro, the gain-of-function effect could be reduced by lacosamide and phenytoin, both commonly used anti-epileptic medications. This indicates that the identification of an underlying SCN3A mutation may have therapeutic consequences in some patients.

What you need to know. SCN3A de novo variants are a novel cause of epileptic encephalopathy. In patients with early-onset epileptic encephalopathies, the functional consequence of these mutations is a gain-of-function effect, resulting in hyperactive Nav1.3 channels that are most likely mediating the network hyperexcitability that results in epileptic encephalopathies. With the discovery of SCN3A as a new cause for epileptic encephalopathies, the Nav1.3 channel joins the other brain-specific sodium channels that are related to human epilepsy.

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Ion channels and brain malformations. When the “channelopathy” concept first emerged – the idea that dysfunction of neuronal ion channels leads to neurological disease including epilepsy – it seemed implausible that such dysfunction could lead to malformations of cortical development. However, recent research has suggested that ion channel dysfunction may indeed be linked with brain malformations. In 2017, we saw convincing evidence that germline de novo variants in GRIN2B can cause malformations of cortical development. Some suggestive, but less conclusive, evidence has also linked SCN1A and SCN2A to brain malformations. Now Fry and collaborators demonstrate that de novo pathogenic variants in GRIN1 can also cause significant polymicrogyria, expanding the phenotypic spectrum of GRIN1-related disorders. As a disclaimer, I am also a co-author on the publication by Fry and collaborators.

Clustering of GRIN1 variants. A schematic diagram of the GRIN1 protein, showing the localization of polymicrogyria (PMG) associated GRIN1 variants on the top in red and non-PMG associated GRIN1 variants on the bottom in yellow. PMG-associated variants cluster in the M3 and S2 regions of the protein, which are important in channel gating and glycine binding. Over 50% of non-PMG associated variants are located in the transmembrane M4 helix, where no PMG-associated variants have been reported. Some GRIN1 variants have been reported in both patients with and without polymicrogyria. However, these patients have only had CT scans, which is not capable of detecting polymicrogyria.

GRIN1-related polymicrogyria. Fry and collaborators have identified de novo pathogenic variants in GRIN1 in 11 individuals with significant malformations of cortical development and associated neurodevelopmental disorders. MRI features included extensive bilateral cortical malformations, most consistent with polymicrogyria primarily in the frontal and parietal regions. One of the de novo GRIN1 variants was identified in a 22-week gestational age male fetus, with abnormal thinning and sulcation of the cerebral cortex, hypoplastic corpus callosum, and ventriculomegaly. This suggests that GRIN1-related cortical malformations may be detectable prenatally.

Gain-of-function vs. loss-of-function. Functional analysis polymicrogyria-associated GRIN1 variants identified an increased sensitivity to NMDA receptor agonists glycine and glutamate, consistent with a strong gain-of-function effect. This is in stark contrast to previously reported GRIN1 variants in patients without polymicrogyria, which were found to have significant dominant-negative and loss-of-function properties. Although this gain-of-function vs. loss-of-function dichotomy may be an oversimplification, it does suggest a correlation between the functional consequence of the GRIN1 variant and the resulting phenotype. Over-activation of the NMDA receptor may result in malformations of cortical development while loss of NMDA receptor function will result in a neurodevelopmental disorder without brain malformation. Although the mechanism whereby NMDA receptor gain-of-function results in neuronal migration defects is still not understood, one hypothesis is that the excitotoxic effects of NMDA receptor hyperactivation may lead to cell death during fetal brain development, which could lead to migrational defects in developing neurons.

Genotype-phenotype correlations. Comparison of GRIN1 variants in patients with polymicrogyria compared to patients without polymicrogyria showed that these variants cluster in different parts of the protein. The polymicrogyria-associated GRIN1 variants were highly clustered in the S2 domain and adjacent M3 helix regions of the GRIN1 protein. The S2 domain forms part of the glycine-binding domain. Glycine is an NMDA receptor activator. The polymicrogyria-associated variants in the M3 region were in a motif known to control NMDA receptor gating. This S2/M3 clustering of polymicrogyria-associated variants in GRIN1 is similar to what is seen in GRIN2B. In contrast, over 50% of previously reported GRIN1 variants in people without polymicrogyria are located in the M4 segment, where no polymicrogyria-associated variants were found.

Implications for precision medicine. It is likely too early to say whether or not these findings will directly influence precision medicine decisions for families affected by GRIN1-related disorders. However, the suggestion of genotype-phenotype correlations does provide additional evidence that may help in a clinical context. This research suggests that gain-of-function variants are most likely associated with a brain malformation phenotype whereas individuals with GRIN1-related disorders without brain malformations are more likely to have loss-of-function variants. Memantine, which is an NMDA receptor blocker, would theoretically be more useful for people with a confirmed gain-of-function variant. However, additional research is needed to investigate the usefulness of memantine in a clinical context, particularly for patients who have an underlying brain malformation.

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Protocadherins. PCDH19-related epilepsy is the second most common genetic epilepsy, behind Dravet syndrome. PCDH19-related epilepsies display the unusual X-linked inheritance pattern in which heterozygous females are affected but hemizygous males are unaffected. Similarly, somatic mosaic males have also been reported. PCDH19 encodes protocadherin 19, a calcium-dependent cell-cell adhesion molecule that is highly expressed in the central nervous system. The long-hypothesized pathomechanism has been cellular interference, although experimental support has so far been lacking. Now, Pederick and collaborators provide evidence that supports the cellular interference mechanism in PCDH19-related epilepsies, bringing us closer to understanding the biology of this unusual genetic epilepsy.

PCDH19 mechanism. The differences in cell adhesion affinities lead to cellular interference in people with PCDH19-related epilepsies. In unaffected individuals (top panel), all cells in the developing brain have the same adhesion properties, leading to proper cell sorting and cell connections. In people with PCDH19-related epilepsy (middle panel), two cell populations exist: cells with wildtype PCDH19 and cells with variant PCDH19. These cells have different adhesion properties and therefore do not sort properly. Proper connections are not are not formed between the two cell populations. In males who carry a PCDH19 variant (bottom panel), a single homogeneous cell population exists. All of these cells have the same adhesion properties and therefore are sorted properly and have proper connections.

Cellular interference. The theory behind cellular interference is that two distinct cell populations exist in the developing brain of people with PCDH19-related epilepsies: cells containing unaltered PCDH19 and cells containing variant PCDH19. These two unique cell populations do not form proper connections with one another, leading to disrupted networks within the brain. However, in hemizygous males who only have one copy of PCDH19, a homogeneous cell population exists. Even though these cells carry a variant version of PCDH19, the fact that only a single cell population exists leads to the formation of proper connections. Therefore the existence of two types of cells – and not the PCDH19 variant itself per se – has been proposed to be the main driver of the phenotype in PCDH19-related epilepsies.

Experimental evidence for the mechanism. Protocadherins, including PCDH19, are adhesive molecules sticking out from the cell surface and can be conceptualized as the tiny hooks and loops of a strip of Velcro. PCDH19 is expressed in the brain with other types of protocadherins, and the various combinations of protocadherins determine a cell’s adhesive properties and thus how it is sorted during cortical development. Cells with similar adhesive properties are sorted together. Pathogenic variants in PCDH19 result in loss of adhesive function, which alters the types of cells the cell adheres to and the way it will be sorted during brain development.

Pederick and colleagues discovered that the brains of female mice who only had one functional copy of PCDH19 – equivalent to people with PCDH19-related epilepsy – showed distinct cell populations that were unable to adhere properly to one another. The cells expressing wildtype PCDH19 had normal adhesive properties, whereas the cells without PCDH19 had altered adhesive properties. The presence of two different adhesion affinities led to missorting of cells during brain development. This abnormality was not seen in male mice who completely lacked PCDH19. In the brains of male mice completely lacking PCDH19, neurons sorted appropriately because all cells exhibited the same adhesion affinities. The authors expand their study by further describing abnormal cortical sulcation patterns in four female patients with PCDH19-epilepsy, suggesting that subtle brain malformations may be a feature of this missorting process. However, due to random patterns of X-inactivation, the severity and presentations in people are likely highly variable.

What you need to know. Pederick and colleagues report evidence from mouse models of PCDH19-epilepsy that supports the cellular interference model. They demonstrate that the heterozygous cell populations – cells containing wildtype PCDH19 and cells containing altered PCDH19 – segregate abnormally during brain development. This alteration is not seen in male mice completely lacking PCDH19, which is consistent with what we see in humans. The authors further suggest that subtle brain malformations may be a part of the PCDH19 phenotype as an extreme consequence of this missorting process.

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