The gold standard. We have discussed various publications relating to trio exome sequencing in intellectual disability, autism, schizophrenia and epilepsy over the last 12 months. The identification of de novo mutations by comparing exome data of patients and parents has become an established technology that has potential both as a research tool and –in the future- as a diagnostic test. However, only a fraction of the mutations found by trio exome sequencing are pathogenic, as roughly 1-2 de novo mutations are also observed in control trios. While it is tempting to think that each and every patient with an unexplained epilepsy must have a pathogenic de novo mutation, the evidence for this is only emerging at a slow rate.

SCN1A, CDKL5. Veeramah and colleagues have performed exome sequencing in 10 parent-offspring trios with therapy-resistant epileptic encephalopathies. They find de novo mutations in 9/10 probands. In 7/10 probands, these de novo mutations include known or presumable epilepsy genes. In particular, two patients with the clinical suspicion of Dravet Syndrome had mutations in SCN1A and one female patient with infantile spasms progressing to multifocal seizures had a de novo mutation in CDKL5, a clinical and genetic finding consistent with CDKL5 encephalopathy. One patient had a mutation in EEF1A2, a gene previously identified in a patient with severe intellectual disability and epilepsy. Three additional patients had mutations in plausible candidate genes. A patient with microcephaly, corpus callosum hypoplasia and epilepsy with complex partial seizures had a de novo mutation in CLCN4, coding for a CL/H+ exchanger highly expressed in the CNS. This gene is from the same family as CLCN2, which has previously been described in idiopathic generalized epilepsies. A patient with generalized tonic-clonic seizures, particularly in the setting of illnesses had a mutation in KCNH5, a gene coding for a potassium channel expressed in the CNS. Interestingly, the patient’s development was normal until the age of 6 months and the phenotypic description including hemiclonic seizures and the generalized EEG discharges are reminiscent of Dravet Syndrome. Finally, a patient with mild developmental delay and refractory complex partial seizures had a mutation in ARHGEF15, a gene coding for Ephexin 5, a protein involved in synapse development. In summary, the findings range from mutations in known genes to recurrent genes to promising genes found for the first time. This motivated me to come up with a draft classification scheme for epilepsy-related de novo mutations.

A classification scheme for epilepsy-related de novo mutations. I am going out on a limb here, but this would be one possibility to classify de novo mutations in patients with epilepsy based on the experience we have had over the last 12 months. Please note that this tentative classification does not take into account prediction tools that tell us something about the severity of the mutations. It only considers the identity of the gene and qualifies the evidence of this gene to be involved in the epilepsy phenotype. This should involve all mutations that are not synonymous, i.e. every mutation that alters the amino acid sequence or splicing. Briefly, Class 1 mutations should be de novo mutations in known and established epilepsy genes including SCN1A, CDKL5 or ARX. There is sufficient evidence that de novo mutations in these genes are involved in the epilepsy phenotype. Also mutations in novel genes where recurrent de novo events have been identified in similar phenotypes should be Class 1 mutations. Class 2 mutations are in genes that were previously described in other neurodevelopmental disorders such as intellectual disability. As it is not immediately clear why mutations in the same gene may cause different neurodevelopmental phenotypes, these genes must be approached with a certain degree of caution in the same way as de novo copy number variations with a broad phenotypic range. Also, these variants might be benign mutation hotspots, but the available control cohorts are still too small. Class 3 mutations are de novo mutations in “promising” genes including many previously not described ion channel genes expressed in the CNS and genes implicated in neuronal development. Everything else is Class 4 and of uncertain significance. Non-pathogenic variants are de novo mutations in genes that were previously found in controls. While one could still argue that these mutations confer a certain risk to disease or might be disease-related if they involve a certain region of the gene, this evidence will be very hard to establish. Please let me know what you think of this classification. I have added an additional category for de novo mutations in established epilepsy genes that appear as individual findings in existing databases (1u for “uncertain”). The significance of these variants is not immediately clear, as discussed in an earlier post.

A proposed classification scheme for epilepsy-related de novo mutations. Not every de novo mutation found in a patient with epilepsy is necessarily pathogenic. I have tried to classify the mutations based on the evidence relating to the gene involved. This classification does not take into account the severity of the mutation. Also evidence in animal models is not considered direct evidence for a role of this gene in human epilepsy. I would be happy to discuss the idea behind this classification and I am looking forward to your input.

The RES working group on unclassified EEs. Reading the paper by Veeramah and colleagues remind me of the RES working group on unclassified epilepsies, where our initial goal was to delineate novel phenotypes either through clustering of clinical features or through genetic studies. I would like to use this opportunity to remind everybody that we might discuss putting together a cohort of patients with unclassified epileptic encephalopathies for trio exome sequencing in the near future. I would refer to the next RES unclassified working group teleconference for this and please let us know if you are not included yet and would like to participate. In summary, with genetic screening technologies available at a much more rapid pace, we might be looking towards an interesting era where phenotypes will emerge based on an interesting interplay of clinical and genetic studies.

Since we saw the geneticist in July 2012, I’ve been expecting Lil Z to eventually get a diagnosis of a-typical Rett Syndrome. The geneticist seemed to strongly believe that was what was causing her problems. And Lil Z checked nearly every single box when it came to a clinical diagnosis of CDKL5:

• Epileptic seizures starting in the first five months of life  - YES
• Infantile spasms – YES (well, flexor spasms, which are very similar)
• Many different types of epilepsy usually including myoclonic jerks – YES
• A small head (microcephaly) – YES
• Hand wringing movements or mouthing of the hands – YES
• Marked developmental delay – YES
• Limited or absent speech – YES
• Hypersensitivity to touch, for example dislike of hair brushing – YES
• Lack of eye contact or poor eye contact - YES
• Gastro‐esophageal reflux – YES
• Constipation – YES
• Small, cold feet – YES
• Breathing irregularities such as hyperventilation – NO
• Grinding of the teeth – YES
• Episodes of laughing or crying for no reason – YES
• Low/Poor muscle tone – YES
• Very limited hand skills – YES
• Some autistic‐like tendencies – Not sure what this means exactly, but probably YES
• Scoliosis – YES
• Cortical Visual Impairment (CVI) – YES
• Apraxia – Not sure what this is, so probably NO…
• Eating/drinking challenges – YES
• Interruptive sleep – YES
• Characteristics such as a sideways glance, and habit of crossing legs – YES

I’d done my research on CDKL5 and Rett Syndrome, as had QB and Lil Z’s former and current nannies and we all agreed – that’s Lil Z. That must be her diagnosis.

So, you can imagine my surprise when I got a call from her Paediatrician yesterday telling me he had tracked down her results and there were no CDKL5 abnormalities reported. So, that’s not it after all.

And we’re no closer to a diagnosis than we were 8 months ago.

As absurd as it is to be disappointed that your child doesn’t have a rare and severe genetic syndrome, I am. I can’t explain it or rationalise it,but I really want a diagnosis for her.

So, it looks like we’re back to the geneticist for more ideas.

I realised that I left everyone with a bit of a cliffhanger yesterday. Would the paediatrician declare that Lil Z has CDKL5 or not?

Unfortunately, he couldn’t give me an answer. He checked the computer records and there is nothing there. But, he said, they often submit their reports in hard copy, rather than electronically (at the moment I’m beginning to think they have also dispatched it from Perth by carrier pigeon). However, because the paediatrician isn’t the one who actually requested the test – her neurologist did, for no reason other than I saw the neurologist shortly after we had our genetics appointment. So, the report would go to the Neuro instead of the Paed.

To make it even more complicated, our Neuro has been off on sick leave. So, the report may be laying unopened somewhere. Bizarre to think information so important to us could be simply sitting in an Inbox somewhere.

Fortunately, he understands that this is important to us and that we’ve been patiently waiting for a long time for the results. So, the paediatrician is going to phone the laboratory to find out if they have completed the test yet – and if so, what is the result. This is likely to take a few days, though, so we might hear back next week.

So it looks like we’ll be hanging over that cliff just a little bit longer…

Watching Peppa Pig

Tomorrow Lil Z has an appointment with her paediatrician. Its nothing special – she has 3-monthly check-ups with him – but I am hoping that he may have received the results of the genetics test for CDKL5. I was told that it can take up to 6 months to get the results, and it has been over 6 months by now. And I want to know. I want a diagnosis

The paediatrician is less enthusiastic about finding a name Lil Z’s syndrome. He is the one who diagnosed her with a genetic syndrome and he’s since been backed up by the geneticist. He was also the first one to refer Lil Z for the CGH array and to test her for Rett’s Syndrome. When all those tests came back negative, he seemed to have lost interest in finding a specific cause for Lil Z’s problems.

This isn’t as dramatic as it sounds. He is still very much interested in treating Lil Z and over the past two years, I think he really cares about her. But, as he repeatedly tells me, a diagnosis won’t make a difference to him. He is treating Lil Z, not a named syndrome.

Lil Z’s therapists tell me the same thing. Whether she’s diagnosed with CDKL5, something else or nothing at all, they’re going to continue the same plan. A diagnosis makes no difference to how they treat her, how they plan to help her development. And that is a good thing. They’re seeing the real Lil Z and not a diagnosis that doesn’t begin to describe her.

So, why do I need to know?

I’ve given this a great deal of thought lately, and I’m still not sure if I really understand why.

Part of it, certainly, is that a diagnosis gives you some indication of a prognosis. At the moment, I don’t know if she will ever walk, talk, use her hands in any meaningful way. I don’t know if she will ever manage to move from PEG feeds back to eating orally. I don’t know if she will ever outgrow her seizures. I don’t know if she will survive childhood – something I don’t like to think about, but a grim reality for many severely disabled children – or if she will outlive QB and me. I don’t know what to expect in the next 6 months, let alone five, ten or twenty years from now.

And prognosis is important. When you have a “normal” child, you know what to expect, what the next stage will be. Vegemite moved from one stage to the next from the time she was born with blessedly boring predictability. It was as if she was reading one of those “Your Baby in the First Year” books at night when we thought she was sleeping. Holding her head up? Check. Smiling? Check. Sitting? Check. Walking? Check. Talking? Check. Meanwhile, I’m still waiting for Lil Z to move off the first page of that book…

A diagnosis also makes it easier to explain Lil Z in 20 words or less. For the past 6 months, Lil Z has had on her hospital chart that she has A-typical variant Rett Syndrome. It hasn’t changed her care in the slightest, but while most of the world hasn’t heard of Rett Syndrome, it does mean something to people in the medical world. And that spares me having to run through the usual list of things that are wrong with Lil Z or that she can’t do.

Also, because our family and many of our friends live overseas, having a diagnosis can help them to find out more and get a better idea of what Lil Z is doing, why she’s doing it, and what we’re going through as a family. Even CDKL5, as rare as it is, has loads of websites, resources and blogs online.

And finally, a diagnosis will give us a way to fit in. That sounds crazy, I know, but so much of the support – and in Australia even the funding – for special needs is broken down into different diagnoses. There are support groups for Downs Syndrome, ASD, Retts, Cerebral Palsy, Epilepsy – you name it, there’s a support group for it. If not an actual one, then a virtual one. But although Lil Z ticks nearly all the boxes for Rett’s Syndrome and all the boxes for CDKL5, without a diagnosis, I wouldn’t feel like I belonged in one of their support groups.

There is even considerable support for SWANs (syndromes without a name). I belong to a Facebook SWAN group but have never felt that I completely fit in – probably because it is based in the UK, so despite sharing many of the problems, the healthcare system is different (which matters when so much of what you’re discussing involves medical treatment and services).

Wanting to fit into a “group” isn’t all about me, either. I want to meet other parents with children like Lil Z so I can get an idea about the future. Not just prognosis, but also what therapies, equipment, medications and who knows what else might help.

My need for a diagnosis is more emotional than medical. But then I suppose, like her doctors and therapists, QB and I have come to terms over these past two years with who Lil Z is. A diagnosis isn’t going to change who she is or what she means to us. But a diagnosis will give us a better understanding of her and potentially new ways to help her.

So, here I sit, the night before her appointment, hoping that tomorrow I’m told that she indeed has CDKL5. No one wants their child to have CDKL5. But at least it would put to rest our search for a diagnosis.

Please take the time to check out the Hope4Harper website. Share it with others. Donate to Harper’s cause. Donate for hope for Harper, others with CDKL5, and those who suffer from epilepsy. 

Merry Christmas, Howard family! We love you! 

The big picture. We have written several posts on trio exome sequencing in various neurodevelopmental disorders this year including autism, schizophrenia and intellectual disability. All studies find a significant burden of de novo mutations in trios, i.e. mutations that arise in the proband but that are not present in either parent. Many of these studies have difficulties interpreting their findings as mutations also occur at approximately the same rate in unaffected individuals. Some studies find a slight increase in patients with neurodevelopmental disorders, but these differences are not as clear-cut as one might expect. This basically means that an identified de novo mutation needs additional information to be considered pathogenic or not. This additional information is either derived from prior knowledge or through pure statistics. Prior knowledge means that this gene is known to cause the particular disorder. Statistics comes into play when a given gene occurs more than once in given a disease, a finding that is unlikely to occur by chance. In all trio exome sequencing studies on autism, for example, interpretation of findings is largely limited to a handful of recurrent genes, as most genes are so-called “single hits” that are difficult to interpret. This scenario is completely different in IS and LGS.

The genetic architecture. Let’s briefly review what we know regarding the genetic architecture of IS and LGS. Both disorders are epileptic encephalopathies and can be caused by a variety of different causes ranging from perinatal brain damage, brain malformations or neurometabolic disorders. Also, in patients with Infantile Spasms, Tuberous Sclerosis appears to be one of the more common causes. However, none of the potential causative factors is present in a significant subset of patients. These patients have IS or LGS for no apparent cause. Mutations in ARX, CDKL5, STXBP1, SPTAN1 and many other genes have been described, but their frequency in patients with epileptic encephalopathies is not known. There appears to be an important role for copy number variations, which are strong risk factors in 5-10% of cases. This leaves us with > 90% of cases with presumably genetic epileptic encephalopathies to be explained.

The Epi4K study. Epi4K, funded by the National Institutes of Health, is a large consortium on epilepsy genetics and the presentation at the meeting of the American Epilepsy Society was the first presentation of data generated by the Epi4K researchers. They analysed 165 patients with IS and LGS collected by the Epilepsy Phenome/Genome Project (EPGP) for de novo mutations that were not present in the parents using exome sequencing. Using this paradigm, the Epi4K researchers identified possibly pathogenic de novo mutations in at least 15% of patients. The list of genes includes many known genes for epileptic encephalopathies, several “single hits” that require further studies and a few genes, which appear twice in this patient cohort. In order to put the data of the mutations identified in their cohort in context, the researchers went on to assess whether these genes are “mutation-intolerant”. This method basically queries large databases such as the Exome Variant Server regarding the known mutations in large cohorts. For example, a gene with many disruptive mutations would be considered relatively mutation-tolerant, whereas a gene without any disruptive mutation would be mutation-intolerant. The mutation spectrum and frequency of a group of genes can then be compared to the data in controls. Applying this method, the Epi4K researchers find that the genes mutated in IS/LGS are usually resistant to mutations, which is another hint at a pathogenic role of these genes.

The Epi4K study. 165 patient-parent trios with Infantile Spasms and Lennox-Gastaut Syndrome were subjected to exome sequencing. The researchers found many known epilepsy genes, several single hits and some double hits. In summary, their data explains 15% of cases with IS/LGS. Taken together, 25% of cases can be explained through CNVs of single-gene mutations.

Paradigm shifts. Even though de novo mutations in IS/LGS have been described in individual cases, the Epi4K data is the first report on a large cohort of patients that has been systematically screened for de novo mutations on a genome-wide level. There are least three paradigm shifts initiated by this data.

First, de novo matters. De novo mutations contribute significantly to the genetic architecture of IS/LGS. While this statement might be trivial for many scientists, I still consider this a surprise. Only two years ago, I would not have thought that de novo mutations would have such a large impact. To me, Dravet Syndrome with the high frequency of de novo mutations in SCN1A was a singularity, an anomaly amongst a highly complex and probably impenetrable genetic architecture. I find myself surprised and relieved that some of the genetic puzzle that underlies human epilepsy appears to be solvable.

Secondly, the known knowns. The Epi4K researchers find a high frequency of known epilepsy genes. Looking back at the last decade of large-scale genomic studies, finding known genes using hypothesis-free methods is the exception, not the rule. It is reassuring that the genes that we have worked on in the past are in fact relevant. Again, this is not trivial as there is no guarantee that candidate genes detected in a small group of patients through candidate or family approaches are relevant on a population level.

Third, the negative findings. It is important to note what the Epi4K researchers did not find. There is no single gene for IS or LGS, but the genetic architecture is heterogeneous. In parallel with the findings in trio studies in autism, it can be estimated that 400-500 genes will contribute to IS/LGS. This will have profound implications for the way we will approach these disorders in clinical practice. Looking for single candidate genes will likely be futile; gene panels or even clinical exome sequencing will be more efficient. Also, the Epi4K researchers did not identify a significant burden of recessive mutations in their patient sample. Even though most patients with IS/LGS are screened for metabolic disorders, I am surprised that there is not a sizeable subgroup of patients with “hidden metabolic disorders”.

Looking forward. The Epi4K data has established de novo mutations as a common feature in IS and LGS. Taken together with the role of CNVs, we can expect that ~25% of the genetic burden of IS/LGS is explained. This leaves us with a large proportion of cases without positive genetic findings. While the rate of pathogenic de novo mutations might currently be underestimated given the unknown role of various single hits, it is unlikely that the genetic etiology is explained by this mechanism in the majority of patients. Even though our previous post on phantom heritability has suggested that we should never aim for the 100% mark in genetic studies, this data begs the question what else is out there as causative genetic variants. Either way, the Epi4K data, explaining 15% of the genetic morbidity at once, is a quantum leap in epilepsy genetics.

Since Lil Z was 6 months and referred back to a paediatrician for assessment, I’ve been reminded constantly what is wrong with my daughter. She has global developmental delay, she has seizures, her head isn’t growing at the rate it should, her brain isn’t developing as it should, she has a visual impairment, she is socially and cognitively delayed, she has no purposeful hand movements, she has no protective reflex, she has poor core strength, she struggles to track and follow, she is unable or unwilling to bear weight through her hands or feet, she has silent aspiration, she is unable to eat lumpy or solid food, she obsessively wrings her hands and scratches at her ears, and she has dysmorphic features.

I know all these descriptions are accurate and necessary when writing up her medical reports. However, she’s still my baby, I love her and I don’t like to see her reduced to nothing but a list of problems. Imagine if parents of a “normal” child were subjected to this when visiting the doctor:

Well, Mrs Jones, we’ve given your son a check-up and feel obliged to point out that he’s a bit slow to walk, isn’t he? I bet all his little friends are walking, but he’s still content to crawl. It might indicate he’s lazy. You might want to consider doing something about that.  And I noticed he cried a lot when I gave him the injection. Obviously he’s a bit of a Mummy’s boy, might grow up unable to cope in the real world, probably never form a meaningful relationship. I can’t really understand what he’s saying yet. Granted he’s only one, but that could be a problem in the future if he doesn’t learn to enunciate better. And his facial features, well, let’s be honest, he’s not going to be the best looking boy on the block. 

OK, so that may be a bit of an exaggeration, but the point is that no one likes to hear negative things about their child. And when you have a special needs child, it seems like that is often all you hear. Which is why I enjoyed the letter from the geneticist, who of course detailed her medical issues, but also commented on her long and crazy hair, the fact she’s able to roll and smile, and said that her gross motor skills were better than he was expecting. He also suggested that we set a long-term goal of helping her to walk. And he declared that her facial appearance was “unremarkable” – in other words not dysmorphic at all. Although I know nothing will change that Lil Z is absolutely adorable, dysmorphic or not, it was nice to actually remove that awful label from the list.

To be honest, I wasn’t expecting much of an outcome from the appointment. I figured he would ask us a lot of questions (which he did), examine Lil Z (which he did), and recommend some further tests (yup, he did that, too). But what I wasn’t expecting was for us to walk out of the office with a likely (although not confirmed) diagnosis.

But we did. And once again, Rett Syndrome is back on the radar.

I feel like I’ve had a love-hate relationship with Rett Syndrome. I’d never heard of it until it was raised during one of Lil Z’s hospital admissions. I had a sneaky look at Wikipedia on my phone and promptly burst into tears. I’d never thought of the possibility of Lil Z not being able to talk or walk, of regression, or that her condition could limit the length of her life. So, Rett Syndrome became my greatest fear.

Still, I’d nearly come to terms with it when the test for it came back negative. At that point we’d had a CGH Array test with no positive results, so QB and I began to prepare ourselves mentally for Lil Z living her life as a SWAN – syndrome without a diagnosis. QB thought she’d never be diagnosed whereas I thought we would eventually get a diagnosis, but that it would be something so obscure that it would essentially be meaningless.

And then we had an appointment with the geneticist. And Rett Syndrome – albeit a rare variant of Rett, called CDKL5 – reappeared in our vocabulary. Its not confirmed – she will need a blood test for that – but the geneticist seemed fairly confident that is what it will turn out to be. We have to jump through some hoops to get the blood test done on Medicare (since its unlikely our private insurance will fund it) so we will be in “probably but not certainly” limbo for awhile.

That said, a diagnosis of Rett’s wouldn’t be a bad outcome. Most people have never heard of it, but it seems to be well-known in the medical community. There are international awareness organizations, support groups, research studies, and even an Australian register for girls with Rett’s.  And, as QB quipped, with a name like CDKL5, Lil Z obviously has the Mercedes of the syndromes…

Its probably good that we’ve had to wait nearly a year for an appointment with the geneticist. Back when Rett Syndrome first came on the radar and reduced me to tears, I wasn’t ready for the news. There was still too much to process mentally about Lil Z and the outlook for her life. Now, both QB and I have come to terms (at least partially) to the fact we have a special needs daughter. And we both understand that Lil Z will be Lil Z, whether she’s a SWAN or has Retts or CDKL5 or some other named or unnamed genetic malformation. She’s going to grow and develop how she’s meant to, with or without a diagnosis.

Harper Howard (credit: hope4harper.blogspot.com)

CARROLLTON (CBSDFW.COM) – She’s nearly a toddler, but she can’t talk, can’t walk and can’t crawl, and for the parents of Harper Howard, it’s absolutely heartbreaking.

“She’s a 17-month-old child trapped in a two-month-old child’s body,” explained Penny Howard, Harper’s mother.

Harper has a rare genetic disorder. So  rare, it doesn’t have a name. It’s simply called CDKL5, which is the gene affected by the disorder.

You can imagine how frustrating it was for the Carrollton couple when doctors had trouble diagnosing the disorder.

“When you have six months of testing and you have nothing, it’s frustrating and disheartening,” recalled Harper’s father, Dustin Howard

CDKL5 affects about 300 people worldwide. It causes intense seizures and spasms that severely impact Harper’s mental and physical development. In some cases, it can be fatal.

[worldnow id=6340848 width=420 height=278 type=video]

“It’s very hard, because everyday you wonder if she’s going to still be with you,” Harper’s mother said with tears in her eyes. “There are kids who have not made it with this condition,” she added.

The family has found a doctor at Children’s Hospital of Boston to study the disorder and to find a treatment. Harper’s parents believe their child could ultimately pave the way to a cure.

“You wonder, why my child? It’s difficult to make peace with the situation that we’re in,” said Dustin. “At the same time we have to believe that she’s here for a reason,” he explained.

Harper’s progress has been slow. She can’t sit up, she’s still fed from a bottle and she needs powerful eyeglasses to see. Her parents say it could take years to perform simple tasks.

But that’s okay with the Howards, because years means there’s still hope for Harper.

“She’s a fighter. I believe she’s going to over come it,” said Penny as she wiped away the tears.

For more information about Harper’s rare condition and how you can help, click here.