#004 - Neonatal Genomics in India: A Conversation with Dr. Shubha Phadke

In this episode of Incubator India, Dr. Akanksha hosts Professor Dr. Shubha Phadke, a pioneer in medical genetics, to discuss the transformative role of Whole Exome Sequencing (WES) in neonatal care. The conversation covers the fundamentals of WES, its clinical applications, the importance of accurate phenotyping, and the utilization of genetic databases for effective diagnosis. Dr. Phadke emphasizes the need for high suspicion of genetic disorders in neonates and the critical role of detailed family history and clinical information in guiding genetic testing. This conversation delves into the critical role of genetics in neonatology, emphasizing the importance of rapid whole genome sequencing for early diagnosis and management of genetic disorders in neonates. It discusses the challenges posed by variants of unknown significance, the necessity of genetic counseling in the NICU, and the ethical dilemmas surrounding genetic testing and prenatal diagnosis in the Indian context. The conversation concludes with a call to empower clinicians with genetic knowledge to enhance patient care.

Transcript

Akanksha (00:03.49)
Hello and welcome back to Incubator India, the podcast where we dive deep into the world of Neontology, from protocols to people, science to stories. I'm Dr. Akanksha, your host for today. And today's episode is very close to my heart, not just because of the topic, but because of the guest who's joining us today. It's an absolute honor and privilege to welcome someone who has been an institution in herself.

Professor Dr. Shubha Phadke, former head of the Department of Medical Genetics at Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow is with us today. She is a name that commands admiration and affection in equal measures. Over a remarkable career spanning decades, she has trained over 700 clinicians through annual national programs in genetics and genetic counseling, established India's

first DM program in medical genetics and led the creation of newborn screening services as well as hemophilia and thalassemia management programs in Uttar Pradesh. She is also a founding president of the Society for Indian Academy of Medical Genetics and part of the editorial boards of leading international journals including the American Journal of Medical Genetics and European Journal of Human Genetics.

With over 300 publications, Dr. Phadke's work has shaped the landscape of genetic diagnosis and research in India. And beyond the clinics and lab, as many of us know her, she is a poetess, an artist, a teacher, and someone who continues to inspire with her grace and humility. Dr. Shubha Phadke recently retired.

but I'm sure the legacy she leaves behind is enormous. As a young faculty in neonatology, I've personally learned so much from her and having her incubated India today is truly a moment of joy for me. And I hope for you, dear listeners, as well. So without any further ado, Dr. Shubha, welcome to the show. We are so happy to have you here. Would you like to say hello to our listeners?

Shubha Phadke (02:29.407)
Hello everybody and thank you Dr. Akanksha for the nice words. It has been really a good journey to see the medical genetics evolve over the world as well as simultaneously in India. And now we know that it has changed the whole diagnosis and treatment of patients of all disorders and especially those presenting in neonatal period. Thank you for having me with you. Thanks again.

Akanksha (02:58.606)
Thank you very much, ma'am. It's a real delight to have you today. So without any further delay, today we are going to unravel something that's fast becoming a game changer in neonatal care, whole exome sequencing or WES as most of us know it. But for many of our listeners, it's still whole exome sequencing or WES might still sound like a high tech distant concept.

So let's start right at the beginning. Ma'am, could you please explain to our listeners what exactly is whole exome sequencing and how is it different from other traditional genetic tests like karyotyping, chromosomal microarray or FISH?

Shubha Phadke (03:45.461)
This is a very important and first point to be clear to the listeners and whosoever is planning to use exome sequencing. We know that the DNA is present in the form of chromosomes what we see. So the whole genome we can see at the microscope level are the chromosomes, but the level of resolution of chromosomes is

very minimal. So when we see one band of chromosome is missing, it will have 20, 30, 40 genes and maybe five or 10 million base pairs. So naturally when it comes to single gene disorders and we want to see one nucleotide change, then the chromosomal analysis is not at all a test to be done.

It can be done only for the disorders like when we know there is gain of one chromosome like Trisomy 21 or loss of one chromosome like Monosomy X that is Turner syndrome. But many of the disorders are single gene disorders and those also present during neonatal period. And then previously the situation was that if you are considering a diagnosis of say maple syrup urine disease.

Akanksha (04:52.034)
Yeah.

Shubha Phadke (05:07.367)
And then you want to find out the DNA mutation or DNA variation, which is causing the disease. You have to sequence all three causative genes and their small segments, one after other, by a laborious time consuming technique known as Sanger sequencing. But now we can have this technique of next generation sequencing, which is a very high throughput technique.

Akanksha (05:27.476)
Yeah.

Shubha Phadke (05:35.989)
and it can sequence the whole genome or many genes in one go and this becomes a very very game changer as you said in diagnostics. And when we say whole exome sequencing, it means that each gene has a coding part and non-coding part. So the coding exons, so if you take coding exons of all genes which we know in the genome and sequence them in one go,

Akanksha (05:36.11)
Yes, ma'am.

Akanksha (05:58.194)
So this is the probably first test we have for video of the day.

Shubha Phadke (06:06.077)
and that is called as whole exome sequencing. So this is a probably first test to be done for many of the genetic disorders whether they present during neonatal period or later childhood.

Akanksha (06:23.978)
Okay, ma'am. Thank you very much. I think you've really simplified it and really tried at your best to make it really understandable, even for those who are not really acquainted or those who are not performing those tests routinely. So next, now that we have gone through and understood what a whole exome sequencing is, when exactly should it be considered in neonates?

And are there any red flags or what are the clinical cues that a neonatologist inside the NICU should be looking for that should raise our suspicion and consider or a neonatologist to consider for these tests in a specific newborn or in a neonate? So what is your take on that, ma'am?

Shubha Phadke (07:17.941)
This is a very important question. Sometimes people tend to say that this is the test for every disorder and every neonate in the ICU. To some extent that is true, but we have to know why we are ordering the test for a particular baby in a particular setting. And at this point I want to clarify. This is a test.

Akanksha (07:30.69)
Yes, yes ma'am.

Shubha Phadke (07:45.673)
where sometimes even if you do not have any clinical clues and like it's a with intellectual disability, the causes can be thousands of genetic disorders. So you can order this test and even if you do not have any clinical diagnosis, you may get diagnosis. Similar situation is there in neonatology because the neonate can only cry or develop ankylopathy, seizures or become comatose. And here,

This is the presentation of all genetic and non-genetic disorders. And many times we really don't know what it is. But we have to suspect genetic disorders in these situations. The red flags would be if there is a previous child affected or not alive and died during neonatal period or present but has seizures or some neurodevelopmental disorder, immunodeficiency.

Akanksha (08:42.446)
or sometimes presence of consensuality. When you place pieces of course, right? The presence of consensuality that is the place where the techniques even survive.

Shubha Phadke (08:43.783)
or sometimes presence of consanguinity even if this is the first child but presence of consanguinity that means the parents are blood relatives even before marriage like it happens cousin marriages uncle these marriages in south india so these are the situations where we think that this could be a genetic disorder sometimes x-link disorders like previous mother's brother died during neonatal period

mother, sister, son died during neonatal period. So this could be suggestive of X-link disorders like ornithine trans carbamylase deficiency. So these are some of the clues, but in addition to that careful clinical examination, careful metabolite profile. So as everybody knows that which are the parameters you look at pH as well as ammonia, sugar and so many things and then there are

these give clues that it could be organic acidinia, could be presence of hypoglycemia which could suggest the possibility of some carbohydrate metabolic abnormalities and so on. So there may be some such metabolic clues as well as clinical presentations like presence of facial dysmorphism, neuroimaging showing cysts in the brain. So there may be such type of clues.

which should be very painstakingly found out. But the most important thing is that to order this particular test, the threshold for suspicion of possibly genetic disorder should be very, very high. And this means that if a good neonatologist feels that probably this may not be an environmental cause like birth asphyxia or septicemia,

And even if there may be some evidence of septicemia, may be also there may be underlying genetic disorders. So very high level of threshold for suspicion of genetic disorders has to be there. And then it comes that as soon as preliminary investigations are done, the exome sequencing preferably should be ordered during the early course of the neonatal illness. One should not wait

Shubha Phadke (11:07.445)
come like TMS and everything and then you suspect and do this test because the test and reporting also may take minimum of 7 to 10 days and hence it should be considered as early stage of neonatal illness.

Akanksha (11:22.51)
Yes, very correctly said ma'am. having said those, keeping in mind those red flags or signs or the history of consanguinity and the prior affection. So those are really some important things to be considered while testing such genetic tests. Otherwise, we may really end up ordering these tests for any and every

a neonatal illness that we are not able to diagnose. So ruling out the commoner conditions and really having still having a high clinical suspicion that yes, this is something that might be genetically related. So then only we need to consider ordering these tests. And once that we have learned about the clinical indications, what does the

Shubha Phadke (12:14.645)
So then I would like to...

Akanksha (12:18.85)
process of whole exomes sequencing looks like on the ground. mean, like what samples do we need? How do we approach consent in such sensitive situations? And how long do the results usually take?

Shubha Phadke (12:33.813)
Before going to this question, again, I would like to highlight one thing that even if there is like say, barcasstrixia is the suspicion of the neonatologist and is a clinical diagnosis, but sometimes it may be barcasstrixia secondary to congenital myopathy. And if there is a history suggesting that the antenatal period was well monitored, perinatal care was very well done.

There was no evidence of some fetal distress during delivery. And in such situation, if there is child presence with neonatal respiratory problems and one label said birth asphyxia. In such situation, probably one has to keep a threshold. Similarly, like if there is septicemia, but in addition, the child had hypoglycemia as well as consanguinity.

Akanksha (13:18.402)
consider.

Shubha Phadke (13:27.559)
then probably one knows that it could be galactosemia and the septicemia may be under superalloyed thing. So again to say that the threshold for ordering this test should be kept minimal. Now coming to the question about ordering the test and giving the sample. This is the easiest thing to be done because it needs only one ml of blood in EBTA vial.

Akanksha (13:41.218)
Yes.

Shubha Phadke (13:56.701)
the most important clinicians responsibility while ordering the test or sending the sample is to provide very accurate clinical information. This means taking the pre-generation family history in a pedigree form. It cannot be just like asking, is everybody fine on your mother's side? Is everybody fine on the paternal side? Asking about consignorate. We are really what it means. It means like

Akanksha (14:05.742)
Absolutely ma'am.

Okay.

Akanksha (14:20.174)
Mm-hmm.

Shubha Phadke (14:25.767)
marriages between cousins or whether your mother and mother-in-law are sisters or something like that. So you have to clarify to the family. Then only you can get correct information. So drawing pedigree is technically easy, but also equally challenging because you will get the correct information if you ask correct questions, especially asking about each and every child of the

Akanksha (14:38.271)
Ready? Go.

Akanksha (14:44.078)
Thank you.

you

Shubha Phadke (14:52.533)
siblings as well as each and every child of the mother and father siblings. So this is very very essential. The second point is providing information about clinical information, anthropometry, there is microcephaly, neuroimaging, clinical examination, certain dysmorphic features. So when we say that many times we are looking for inborn errors of metabolisms when it comes to

babies in NICU. But some of these inborn errors have some dysmorphic features and sometimes there will be microcephaly, the neuroimaging may show cysts and previously some years ago or a decade ago, we used to consider that if neuroimaging shows cysts the brain, most likely it is secondary to the birth astrixia or some perinatal insult. But we know now many

Akanksha (15:39.918)
.

Shubha Phadke (15:50.413)
metabolic disorders like metabolic cofactor deficiency, they may have cystic leaches, pseudotorch syndrome, may be falsely labeled as torched encephalopathy. So it's very important that the detailed clinical examination is done and the whole information is provided to the laboratory. Those who are working in a good centers, they should also go to the website called HPO.

Akanksha (15:59.493)
Hmm.

Akanksha (16:09.23)
Laboratory.

Shubha Phadke (16:20.425)
human phenotype ontology and here all those clinical features and parameters they have given one HPO term and number. So if you can list all those your findings in these HPO terms and HPO numbers and give it to the lab then the possibility of diagnosis correct diagnosis increases because all analysis of NGS based testing whole exam sequencing is computerized.

and in computerized they will feed the sequencing data of the sample and if you can provide your clinical features in HPO terms then those also can be in computerized form and the diagnostic will definitely improves if the clinician provides accurate correct information. Other than that reporting time, is very critical in case of neonatal ICU babies.

Akanksha (17:06.222)
Yes ma'am, absolutely.

Akanksha (17:18.914)
Yeah.

Shubha Phadke (17:19.125)
So earliest would be 7 to 10 days. And now the thing is coming that rapid exome sequencing results within 3 to 4 days. So this is very important because if you get a treatable disorder, it should not be delayed. And this is very important because we see that in case of NISD when you send the sample for say biochemical testing.

Akanksha (17:26.454)
rapid equal yes ma'am

Shubha Phadke (17:45.043)
Many times what happens first one or two days are gone in doing preliminary investigations and the child is put on IV fluids. So even if those diagnosis which can be you can get by TMS or GCMS, many times those parameters are changed or additional findings that you to septicemia or other things also change the metabolic profile. And in that situation,

you may not get a correct diagnosis even if it is a simple diagnosis like maple syrup urine disease because the sample scent is not sent at a appropriate time and now the child is on IV fluid so the whole thing gets changed. In such situation, testing NGAs based testing because it's a DNA based testing and it will not be changed with treatment so it is going to be the same.

Akanksha (18:23.796)
Okay.

Shubha Phadke (18:41.941)
along with other metabolic testing like PMS, DCMS if this exome sequencing is simultaneously sent galactosin. One thing we know that we can do total galactose, we can do gall test. But by the time the patient has, the baby has received blood transfusion, then the gall test cannot be done or it will make the continuous results. So very easily treatable disorders, they also can be picked up.

Akanksha (18:52.782)
Hmm. Hmm.

Shubha Phadke (19:10.165)
by NTS based testing and earliest results more and more labs will start providing this maybe at extra cost but at least four to ten days testing time for results will be useful especially for if you are looking after treatment. Then secondly there is some second group of disorders where

Akanksha (19:24.526)
.

Shubha Phadke (19:37.927)
Diagnosis is very important even if the neonatologist feel that the child is not likely to survive. Parents also have given up and they don't want to spend more time. But here the diagnosis is very important because there may be possibility of recurrence. In that situation, the time to diagnosis and reporting time of injuries is not that critical, but still it has to be same.

especially it appears very sad but sometimes it's a practical situation that the child is not likely to survive and still we should encourage the parents to store the sample or send the test for NGS based exam sequencing or at least store the sample and sometimes getting sample is also very difficult so just few drops of blood on the filter paper

Akanksha (20:16.238)
.

Akanksha (20:25.57)
Yes.

Shubha Phadke (20:33.729)
or even a piece of umbilical cord, just one centimeter piece of umbilical cord, which may not have blood, that also can be good source of DNA. So depending on what you want. So if you are looking for the treatable disorders and immediately want to make change in the management, then very well, one should try to send the sample early and ask for the quick reporting.

Akanksha (20:59.726)
Quick results, yes. Absolutely, ma'am. We really got some learning points from you. And I think that's such a crucial point that you highlighted regarding pedigree. Because we often jump to ordering advanced tests, like whole exome sequencing, without actually first doing the basics, like taking a three-generation family history, as you said, and then drawing the proper pedigree.

It can really offer valuable clues, sometimes even pointing to a likely mode of inheritance, before we have the results of the genetic test with us. So that was really good that you

Shubha Phadke (21:35.285)
This is...

Shubha Phadke (21:39.317)
I can just share one case where the mother, sister's son died during neonatal period and there was a small piece of paper where the ammonia value was written and which was high. now, and there was similarly another, their child also died. So now we know that both, it's very high possibility that X-linked recessive disorder.

Akanksha (21:43.586)
Yes ma'am.

Akanksha (21:55.051)
Okay.

Shubha Phadke (22:08.817)
and maybe urea cycle disorders. So immediately it falls down to ornithine trans carbamylase deficiency. And then not only with none of the children were alive and no sample was available. So we just tested the mother for carrier status of OTC deficiency. And she was carrier of pathogenic variation in her DNA.

Akanksha (22:24.27)
you

Akanksha (22:34.004)
Okay.

Shubha Phadke (22:36.545)
So sometimes it can be such a very important thing can give you a clue and they can be provided information about risk of recurrence and prenatal diagnosis to prevent the recurrence in the family.

Akanksha (22:47.623)
you.

Akanksha (22:51.682)
Yes, yes, absolutely, ma'am. And I think also, as you highlighted, that before we move to interpretation, one more aspect that's often overlooked from our side maybe is how we describe the phenotype. That's equally important when we are ordering these tests. And as you suggested that human phenotype ontology can be like it.

really provides a very structured vocabulary for the phenotypic abnormalities. And instead of using some vague terms, we can use some standardized descriptors like micrognathia or hypotonia. So they can really help the geneticist in doing his analysis more effectively. think that's a, and maybe it.

Shubha Phadke (23:35.381)
Thank

Akanksha (23:43.138)
can also make it like the interpretations much more faster and more accurate if we have made use of such structured vocabularies as you suggested. So that was a very good point taken.

Shubha Phadke (23:59.381)
One more point I want to highlight about phenotyping. Yeah, many times in neonatology we are more thinking about inborn errors of metabolism. But many other genetic disorders which are not like inborn errors of metabolism like something like ontocerebral hypoplasia or there can be sometimes even lysosomal storage disorder like VC syndrome where there is there are coarse faces and then aplasia of corso corpus callosum.

Akanksha (24:02.733)
Yes, ma'am.

Akanksha (24:08.28)
Yes.

Shubha Phadke (24:28.649)
There are many syndromes associated with congenital contractures. these are all also, phenotyping is very, very important and here photographic evaluation, small cornea or long palpated fissures, puffy cheeks, some of these can give clues to the meta-inborn errors of metabolism. So phenotyping documentation by photograph is very, important.

Akanksha (24:33.078)
Okay.

Shubha Phadke (24:58.781)
Neuroimaging as all of you know, it's very important and of course, whenever possible it should be done so that we can get some idea about these are important parts of phenotyping. The other part of phenotyping is called as reverse phenotyping. So we have sent up a sample and we got unpredictable, unexpected result. Maybe sometimes we are also not aware of that particular disorder. It's reported recently or very rarely.

Akanksha (25:14.902)
Okay.

Hm.

Shubha Phadke (25:28.841)
Then one collects the information about that particular disorder, goes back to those original publications, see their photographs. And then after that, we again can examine the child to look for those features, some of which we might have missed. And one such was that the child had epidermal isosyptomia. And they sent us photograph and the samples for some baby was admitted in some other neonatal ICU.

Akanksha (25:33.324)
you

Akanksha (25:49.998)
Okay.

Shubha Phadke (25:58.195)
So we did, we got the test done and there was a single mutation in a gene for this epidermolysis bullosa. But that is a resuscitive disorder. So we told to lab to look for another mutation. They reanalyzed and identified. But that particular gene causes epidermolysis bullosa with hyaluronic stenosis. So when the family came, the husband came and I asked the child was not alive by that time.

Akanksha (26:08.63)
Akanksha (26:22.72)
Okay.

Shubha Phadke (26:28.127)
So I told her whether he was, the baby was vomiting frequently or there was some feature of pyloric stenosis. So to my surprise, he said the child had pyloric stenosis and was operated for pyloric stenosis during the warm period. So one important clue was missed, but luckily after we got the result, we could collect that information and then say that the DNA variation identified in that particular gene.

Akanksha (26:42.923)
Okay.

Hmm.

Shubha Phadke (26:56.585)
which not only has epidermolysis bullosarv, but pilaris stenosis, that was some variation of little less significance or likely pathogenic, because of the phenotype, very specific phenotype, we can change that likely pathogenic to pathogenic. So such type of detailed phenotyping after the result, reverse phenotyping is very important and this will can be done only if you do the review of literature.

Akanksha (27:15.662)
you

Shubha Phadke (27:25.378)
and look into OMIM and other sources of information.

Akanksha (27:29.294)
Yes, ma'am, really very correctly said. Actually, tools are, as you said, OMIM, the online Mendelian inheritance in man. And I think there are some more gene matcher, face to gene. Some of these I am aware of. Would you just maybe just touch upon it?

Shubha Phadke (27:35.029)
you

Shubha Phadke (27:39.317)
you

Thanks again.

Shubha Phadke (27:50.193)
Yeah and the pub made first omim is very important and omim over years sometimes some of the genes are not that updated frequently so omim is a good source and you can get number of genes so even if you have a metabolic profile suggestive of say MSUD.

or propionic acidemia and then you go and find out how many genes are for that particular disorder. So you will get a complete information. The point I would like to tell the listeners is that go scroll down at the bottom and see the date of update. So if it is like 24 something, 2024, then we know that you have almost latest information.

Akanksha (28:32.398)
Okay.

Akanksha (28:39.926)
Latest, yeah.

Shubha Phadke (28:41.311)
But sometimes if it says it is only updated up to 2018, then probably it's better to go to PubMed. looking into the PubMed as well as Google, everything is very important. Not for neonatologists, but there are sources of information like CleanGen or CleanVar where the information about the DNA variations is also available. Of course, it is like...

Akanksha (28:49.132)
Okay.

Shubha Phadke (29:08.685)
These databases, one has to be very well conversant with those use of databases. So they can give information about the DNA variation with whatever you have got in the result, whether it's likely to be disease causing or not. sometimes because the lab reports some DNA variation, which may match with the phenotype, but still may not be disease causing because some variations

If you say see epidermolysis bullosa, there are so many genes that if you test, then you can get some variation in one or two genes or same with cardiomyopathy. So naturally, we have to really be sure whether the DNA variation reported in the report is really harming the gene or really likely to cause the gene will be really causative or not. So the reports mentioned pathogenic or likely pathogenic.

So we had to look at the report as well as one can try to get our own impression to classify that variation to find out. You can go to the database called Franklin. These are some of the things where you can get some more information about the DNA variations. Phase two gene, as you said, put the photograph and then some image analysis and.

Akanksha (30:06.616)
you

Akanksha (30:26.124)
Yes, ma'am. Sure, ma'am.

Shubha Phadke (30:33.446)
It depends. of the phenotypes are so classic that you will get that correct diagnosis within first-stage differential diagnosis. And one can always try all the databases, including Google. Many times you can get something important through Google. But whatever you get the information, one has to verify the source of the information, reliability, as well as then apply it judiciously. You cannot like there.

Akanksha (30:36.888)
Classical yes.

Akanksha (30:47.278)
Hmm.

Shubha Phadke (31:02.899)
The clinician skill or neonatologist skills will be always required and this to make final decision about the diagnosis, correct diagnosis.

Akanksha (31:14.382)
Okay, yeah, point very well taken, ma'am. And actually, I also like you touched upon rapid whole genome sequencing, like wherein we can expect that around the time for the results may be like really less than seven days or maybe three to five days. And I think like there is an increasing role for especially the critical ill neonates wherein early diagnosis can actually change the management dramatically.

Could you just briefly touch upon what are the current availability and limitations of this test in the Indian context?

Shubha Phadke (31:52.469)
Absolutely rapid testing is very important because most of the there are a lot of publications and they say 20 to 40 percent cases you can get some disorder, correct diagnosis and many of them may be treatable. So the whole objective of this exercise to be able to treat the child appropriately and so if the diagnosis comes to be

Akanksha (32:14.67)
Hmm.

Shubha Phadke (32:19.177)
Paradox in responsive seizures so you can make that particular diagnosis But the question as you said that rapid diagnosis is very important But the variation if it is really damaging pathogenic or causative likely pathogenic Or this is one important thing secondly the phenotype should match So if you get a variation and we aware the phenotype is not matching

Akanksha (32:24.472)
Yes.

Akanksha (32:43.266)
Mm-hmm.

Shubha Phadke (32:47.913)
then you have to really consider whether this may be. So you get a variation in crab aging in a neonate who is three day old and very sick. So we know that this may be additional disease because now with this whole exome sequencing, because it's multi-targeted, we are identifying some children, adults with more than one genetic disease. So it's a first thing that the variation you may not get anything.

Akanksha (32:52.596)
you

Shubha Phadke (33:15.603)
you may get the correct diagnosis or you may get some finding which is appears to be very important but it's not matching with the phenotype. So all these exercises has to be done and discussed then something like that so sometimes you get a diagnosis say biotinidase deficiency. The easiest thing is and the phenotype is also matches matching but the variation appears to be

not reported in normal people as well as not in the disease causing patients and these are called variations of unknown significance. So what do you do for that? So in this variations of unknown significance, many of them may not be really disease causing. It's only because they are not reported in the normal population because the databases of normal population may not still does not have much of the

Akanksha (33:58.734)
In your system, you are still able to use this one.

Shubha Phadke (34:12.797)
representation of Indian population. So many of the variations, are not there in those databases of 1000 genome or exact and so many things. And then we say Nomad. Nomad is the biggest database of normal variations in the normal people. But they have very limited coverage of Indian population. So it happens that our way many variations which are

It may be common in Indian normal people, but they are not reported. So in such situation, based on the variations of unknown significance, we usually cannot make use them that for correct diagnosis. But it is something like biotin, it is the efficiency then giving biotin as a trial. And then if the child responds, then we know that yes, it is the very important phenotype criteria that the

child with seizures responded to BARTing. So such type of sometimes you can take actions based on variations of unknown significance but most of the times these are not likely to be positive and their utility has to be you can make further testing later on based on that. So if it is say molybdenum cofactor deficiency gene and the variation is

variation of unknown significance. You look at the detailed brain imaging, you do other sulfide metabolites in the urine and so on. So one can try to work up further to get more information about the gene. If there are multiple affected family members, then you can test them and try to get information if it is segregating with the affected family members and so on.

So this is a very big challenge and this challenge is more because the database of disease causing variations in India as well as huge database of normal population is still limited in Indian situation.

Akanksha (36:24.055)
Yes, yes ma'am. think that's such a powerful point and very real in day-to-day in ICU practice. The report of uncertainty about the variance of uncertain significance, we just get a report and we are left asking what now? And this is, as you said, because in the Indian population where the genomic databases are still evolving,

So definitely it is particularly challenging in the Indian context because the population representation in the global genetic databases is still limited. So as neonatologists, many of us aren't actually trained to interpret or act on these results confidently. So how do we deal with this uncertainty?

Shubha Phadke (37:11.485)
Yeah, so referral to clinical like nowadays all these expertise are so different and that's why we need to have a multi-specialty clinics as well as meetings together once a week to discuss all these and as you always keep on referring such cases to us and that is very important if we can be of any use in some times.

point you said what to do with the variations of unknown significance. So at least if a neonatologist thinks this is a question and doesn't immediately anything and tells us clinical genetic in the area that is very important. Sometimes some people feel that yes I thought of there is acidosis and this thing and we I thought it is organic acidemia and we have got a variation so

phenotype matches and then they immediately decide that that variation of unknown significance is the causative. So I want to make everybody alert for this point. Just organic acidemia is a very very rough diagnosis and naturally saying just based on that non-specific cardiomyopathy. There are so many genes for cardiomyopathy. So

phenotype matches should be very specific as I said epidermolysis bullosa with pyloric stenosis only one causative gene that there you can be very specific that this phenotype matches but otherwise just saying that encephalopathy seizures and then the particular gene for encephalopathy epileptiform encephalopathy also cause similar phenotype.

Akanksha (38:44.078)
Thanks.

Shubha Phadke (39:04.617)
but this is a very non-specific phenotype. There are hundreds of genes which can cause epilepsy during neonatal period and there can be many metabolic disorders. such type of non-specific phenotypes, can show one should not make a cover use to confirm the variation and say that this is likely positive because the phenotype matches. But such phenotypes, vague phenotypes, non-specific phenotypes,

Akanksha (39:14.576)
Yes.

Shubha Phadke (39:33.663)
cannot be used as a criteria to re-classify the variation of unknown significance to likely pathogen. One should be very very cautious while making reclassifying at your own level.

Akanksha (39:49.142)
Okay, ma'am. Thank you for bringing that out. And actually, it automatically takes us to the next segment or very important part of it, the counseling, the genetic counseling. Anyways, what are the key aspects of genetic counseling for families in the NICU once a diagnosis is made or even when it's inconclusive?

As we just talked of the variance of unexplained uncertain significance, how do we counsel parents when we even don't fully know the implications of such tests? Then often we have some incidental or secondary findings. So how do we approach them ethically and practically, especially when they may not be immediately relevant to the neonate's current health? So those are some.

dilemmas that often we may feel explaining to the parents or the family. Can you please explain and elaborate it on a bit?

Shubha Phadke (40:51.445)
Yes, Dr. Akanksha, this is a very powerful technique and naturally because multi-targeted. So you have the, that is its strength where you don't have a diagnosis. can look at thousands of genes in one go and give you correct diagnosis. The second point, the same thing, also leads to, reposes some dilemmas because it will look at many other things which you are not loved.

expecting and finds out something unrelated like some variation in the BRCA1 gene, which is not relevant to the child, but may be relevant to the mother from whom the child maybe has inherited. So there can be such secondary findings. But before we discuss that, I wanted, I forgot to mention that when we want to send the sample talking to the family about

Akanksha (41:28.526)
Hmm.

Akanksha (41:36.046)
Hmm.

Shubha Phadke (41:49.151)
pre-test counseling is very important. And second, we have to tell that this is a multi-targeted technique, very likely to give diagnosis about maybe 20, 30%. But being costly simultaneously in the same breath, have to tell that even if the child has serious disease, it may not be able to give us any positive report because when patient pays for it.

Akanksha (41:51.8)
Yes.

Shubha Phadke (42:14.995)
they feel that if it is costly test, it has to give some positive results. So unfortunately, sometimes it says no pathogenic variation identified. And secondly, we always would advise that while sending the sample in any situation, especially during neonatal new MSU, a triosat testing should be done. So along with the neonate, preferably

samples of father and mother should be sent together so that the interpretation of that variation becomes very very useful. Unless you test the parents simultaneously and compare their exome sequencing results with the results of the exome sequencing of baby, you cannot identify de novo variations for autosomal dominant disorders.

Similarly, if you identify two variations in our gene for autosomal recessive disorder, then we have to know before deciding that it is causative, we have to know that one variation should be on one chromosome or one allele inherited from father and other on the other chromosome, which is inherited from mother. So unless you test both the parents simultaneously.

you will not be able to tell is the whether the both variants are if they are on the same one chromosome or one copy of the gene and other copy of the gene is absolutely does not have variation then the interpretation is that this is not likely to be the cause of disease. So I would not go into details but pre-op testing is very important and we have to tell the family that because we are testing both of them.

Akanksha (43:53.27)
So, I will not go into details, but your testing is very important and you have to tell your family that because you are testing, it will not work.

Shubha Phadke (44:04.597)
Sometimes you may identify some DNA variations which may be giving you idea about serious disorders in you which may present on later on like as I said cancers or other is serious disorders, especially cardiomyopathy and Arrhythmogenic disorders like which are which force the patient or individual for sudden death. So these are the

Some of the important groups of genes which are considered that they may be shared if you get a pathogenic or likely pathogenic variants in such genes, they may be shared with those asymptomatic individuals in the family because it may implicate their health. And this type of counseling is very challenging. Many times at that particular time.

the child is so sick that the family is concerned about the child and so for the time being my personal suggestion is that such type of unrelated variations may be left for the time being not to be shared with the family unless they are directly implicated in the health of that particular neonate. Of course later on the lab and the clinician and then the parents

May discuss and decide about that the type of secondary findings But what is right for our situation and India and what people think is totally impossible to decide So we can just briefly tell them that you may find out some genes for cancers or something Would you like to know and they may say yes or no, so we had carried out survey for lay persons

Akanksha (45:46.638)
Yes.

Shubha Phadke (46:00.017)
And many people said we would like to know everything, but I'm not sure whether they understand what they mean by everything. And it's really challenge. We need to work more on this area to find out what general people feel about it. And but such type of secondary unrelated findings implicated in the health of the child later on or of the parents. Probably we need to briefly tell them that they may be.

Akanksha (46:04.46)
Me. Yes.

Shubha Phadke (46:28.871)
and whether they would like to know or not and that can be communicated to the laboratory. But trio testing has to be done. So this is one important part of pre-test counseling. So after the results, we already have told them that sometimes you cannot identify anything. and this anything and that it doesn't mean that the child does not have disease. has a disease, also maybe genetic disease.

Akanksha (46:37.326)
Yes.

speed test.

Shubha Phadke (46:57.599)
For example, see if the child is very hypotonic and we have said send the sample thinking that it could be glycogen storage disorder, it could be kandirati mahatati. And then, but we haven't tested the mother. when mother, because mother probably had undergone cesarean and the neonatologist did not get to see, but after exam sequencing when they were called and

you talk to them and then find out that mother has myotonic phases and the cause of hypotonia in the child is probably congenital myotonic dystrophy. So this will not make me pick up in your routine exam sequencing because such type of triplet repeat disorders. Now people have some algorithms to even identify them but routine exam sequencing many times

Akanksha (47:34.482)
My

Shubha Phadke (47:54.279)
does not identify some disorders like this one which comes to my mind for neonatology and genital myotonic dystrophy. If you haven't told to look at the mitochondrial genome then some disorders present of mitochondrial genome they may not be picked up. So there can be various reasons that there may be genetic disorder not picked up or if it is even your diagnosis is very definite our diagnosis is say

Akanksha (48:14.478)
you

Shubha Phadke (48:23.957)
some metabolic disorder, but you haven't got any variation because the variation may be deep in intron. So if you remember in the beginning, I told that by exome sequencing, we are sequencing the only coding regions. So in between those coding exons, there are introns and sometimes there may be variations in that intron, which are also damaging and can lead to disease. So

Akanksha (48:28.814)
Mm-hmm.

in trance.

Shubha Phadke (48:51.679)
There are very many reasons that the causative variation may not be identified and sometimes it may be really non genetic disorder or it may be like missed for various reasons. So when we tell the family that such costly test was done and we were expecting great current diagnosis, but we haven't been able to find out. this is also difficult to explain. And for this, can tell them that after one year, probably

Akanksha (49:15.214)
.

Shubha Phadke (49:20.955)
It may be right to look at the reanalysis because sometimes more information is available and then the cause can be identified. The next step to that is that if we have a specific diagnosis in our mind and then we can go for whole genome sequencing and to look at that introns and other parts of that particular gene. So this is again costlier.

Akanksha (49:32.398)
The next step is that we have a specific diagnosis in our mind. And then we can go for full genome sequencing and to look at that, we cross another part of the network if you please. So this is again possible, that I'm holding on to. So this information may have to go right to the family. But the best part is that if you have a new kind of academic positive in the game.

Shubha Phadke (49:49.459)
than whole exome sequencing. This information we have to provide to the family. But the best part is that if you have identified a pathogenic positive variant in a gene with a treatment possible, so that is the best option and then probably the whole thing changes and the whole family like the outcome will improve. Sometimes it is a treatable disorder, but already the time has lapsed.

Akanksha (50:01.4)
Yes.

Shubha Phadke (50:19.349)
the child is very sick or sometimes you have identified a variation in a definite causative mutation but this disease is not treatable and then the family can consider thinking about whether to really continue that in serious in the costly neonatal ICU treatment because the outcome of that particular disorder is usually not correct.

But after all these things, the family many times are very disturbed and they say we just want this child, we will never think of another child. But it is important to mention to them about the possibility of recurrence, especially those autosomal recessive, X-linked recessive disorders and also give it in the summary, type written summary, so that now or later on, and they should be preferably called after three months.

or to discuss about these issues when they are really little acceptable to these situations. So at the same time, the child, you are treating the child or also their parents are grieving about the loss of that child, it's very important that the family should be provided information and maybe later, recall later can be sent so that they can come back and again.

discuss this issue and plan the next pregnancy. I remember many cases seen in your neonatology and the diagnosis was made and they came really at right time before planning the next pregnancy and then they can be provided information about risk of recurrence and then the prenatal diagnosis is an easy option to prevent recurrence of such serious and sometimes untreatable disorders.

Akanksha (51:55.246)
Thank you.

Shubha Phadke (52:15.669)
The challenge is there are many challenges again and I remember a case where your diagnosis was congenital myopathy and the baby had three DNA variations all of them pathogenic and likely pathogenic two in one gene for congenital myopathy and the other in the other gene for congenital myopathy so nimalin myopathy and so we really will never know

which one of them is causing disease or whether all three of them are causing disease. But we had to do prenatal diagnosis for all three variations. Cost was increases, but most important thing is that the child should not have any either of these variations in either of the genes. Otherwise there can be recurrence. then this is again challenging for counseling. We also feel very anxious about such families.

Because when we say autosomal recessive disorder, risk of recurrence is 1 by 4. But that is for only one disease. What will be the risk of recurrence if there are two autosomal recessive diseases in the family and then probably there will be some babies with both the diseases, some with one disease, some with another disease. So all these things, communication of this counseling is very, very challenging and

So, but it provides option of prevention of recurrence by prenatal diagnosis. So, if you identify the positive variation, all end is good for doing all these exercise of exome sequencing.

Akanksha (54:00.83)
Surely, ma'am. Actually, thank you for highlighting the importance of pre-test counseling. It really is the foundation, like really making hap- helping families understand process and then also prepare before they are faced with some complex decisions. And as an NICU, we say that every moment counts, but then taking time to counsel isn't actually a delay. It's an absolutely important act of care.

I must say.

Shubha Phadke (54:34.065)
Important act and that the head of the department and senior consultant and consultant should know that it is their responsibility and not the responsibility of the resident to give such type of news sometimes which are bad news. So it's very important that communication. This is very, very challenging and enough time and everybody should be together so that one can communicate self-information.

Akanksha (54:47.084)
Residents, yes.

Shubha Phadke (55:02.877)
Not only all these exam sequencing, just birth of a baby with child with trisomy 21 or Down syndrome. This is not a very challenging diagnosis, but it's a very, very important moment for the PAP family. And at birth, the diagnosis should be immediately conveyed to the parents as well as the nana, nani, dada, dada, the grandparents if are available so that you can do appropriate testing.

Akanksha (55:09.24)
Absolutely.

Akanksha (55:17.964)
Hmm.

Shubha Phadke (55:29.705)
see that child does not have T. Oficiola, cardiac disease and so on. providing such type of diagnosis, any genetic disorder, and then again complex things like exam sequencing is a very challenge and everybody has to learn communication by talking to the patients and respecting them. So communication is itself a big topic in itself and everybody has to learn that with practice and understanding.

Akanksha (55:53.754)
Yes, yes, ma'am. Absolutely. Yes, just going a step further, ma'am, genetic screening and even prenatal whole exome sequencing is now increasingly becoming the norm in the West. But I think this opens up some complex questions. How relevant is it in the Indian setting?

Is it really ethical to offer such testing when full interpretation and counseling are not always available? And how to really balance these tests with the emotional weight of what might follow? there may be, like it comes to decisions about around pregnancy, termination, or feeling like we are becoming the decision makers for the fetus. So these are really huge dilemmas, I think.

every clinician especially the neurologist and obstetricians need some guidance upon like how to walk this tight rope with empathy and integrity so your words on that man

Shubha Phadke (56:59.505)
Yes, this is a topic very close to my heart because on one hand, is a exam sequencing is a very powerful technique and providing diagnosis when nothing was possible. Sometimes we have just as the parent child is not alive or sometimes parents bring a piece of umbilical dried cord and we have diagnosed SMA based on that DNA extracted from that.

Akanksha (57:16.398)
Hmm.

Akanksha (57:28.451)
Mm.

Shubha Phadke (57:28.511)
So these are all enormous possibilities. But at the same time, there are so many limitations of these type of testing. So these many times are not understood by the policymakers, including I feel that many guidelines which are coming that you can test all couples for thousands of autosomal recessive serious disorders.

and then offer them this prenatal diagnosis so that they can be guaranteed or at least birth of serious genetic disorders can be avoided. But one has to understand that here, pre-vention is always better. But pre-vention here, it means termination of the baby affected with the genetic disorders. So here we have to be...

Akanksha (58:19.086)
Hmm.

Shubha Phadke (58:21.653)
110 % sure that when you are testing and making a diagnosis of foetus and for a serious genetic disorder and then giving option of termination, it has to be 110%. As we said, many variations of unknown significance. So no prenatal diagnosis should be based on that. Secondly, the likely pathogenic variations, some of them may be 10%, maybe more are

probably benign. So for families who have one child with some serious disorder, say a neonate or a fetus who died in neutral, you can test everything to make a correct diagnosis and then provide if they are you identify really causative pathogenic likely pathogenic variant, then provide genetic counseling prenatal diagnosis. But when it comes those families who do not have

Akanksha (59:18.69)
families who do not have any family or any kind of support. And there you all have received.

Shubha Phadke (59:20.677)
any family history of any genetic disorder and then you offer testing for all genetic disorders. So you want to identify whether husband and wife both are carriers of any serious genetic disorder and then tell them that you may have a child with genetic disorder. So we can test the baby in the womb and the baby has a disease then you can terminate and then only when have a

Akanksha (59:36.878)
you

Shubha Phadke (59:49.523)
the baby does not have disease you can have a that you will be normal baby and all these diseases like osteopetrosis, inborn errors of metabolism so many things can be prevented but here the question the whole objective is very good and want to help but you have to understand that when there is no family history and you identify some variation like G-pathogenic you really do not know what will be the whether the child

Akanksha (59:59.662)
you

Shubha Phadke (01:00:18.645)
those variations from mother and father will really have a disease or not as some of them may be harmless. And secondly, what will be the cause? We have seen children with crabby disease. As you know, crabby disease manifest as two, three, four months and they would die during infancy or at the most one or two years. But we have seen crabby disease presenting at 15 years of age. There are case reports where the disease

presented at 40 years of age. one, so much of uncertainty is there. So, though exome sequencing, whole-g-clone sequencing, we can sequence each and every nucleotide, but the inference of those nucleotide changes, there is a lot of limitations because we don't know a lot and we also do not know like that sequence variation, which may be disease causing in a family.

Akanksha (01:00:57.518)
you

Shubha Phadke (01:01:16.349)
in other family with their other genetic background, can it be harmless as well? So this type of over enthusiastic approach of testing all families without families for carrier detection of rare genetic disorders, which are supported by guidelines of some countries and also maybe the...

Akanksha (01:01:33.687)
Thank

Akanksha (01:01:39.982)
Hmm.

Shubha Phadke (01:01:43.227)
marketing aspects of laboratories, are sort of, it appears very, very logical that you can guarantee the child normal baby to the family. But the hidden behind is that you may be terminating some normal babies and then prevent the families from having such normal. If they have one child with again, fetus affected, you terminate. Second also may be affected, they terminate.

Can you understand the enormous emotional trauma which the family will undergo? There may be some interpersonal relationship between husband and wife. So it is too early to provide such type of carrier screening to families without family history. So if somebody wants to help the couples, take three generation family history. If there is some family member in the family with sedition muscular dystrophy,

or a sudden death or something, then you can appropriate a council and get appropriate testing for those disorders. offering testing for all genetic disorder is probably something over enthusiastic thing, which we may be causing harm to many, families in different ways. And most importantly, many normal babies may get terminated.

So this is a very important message for everybody. It's not in Indian settings. India, everybody has access to state of art, healthcare, as would be there in the developed countries. But the issues remain the same for even international setup. think they have to really think, and many European countries and some other countries, they are not much favorable for.

Akanksha (01:03:17.199)
you

Shubha Phadke (01:03:37.097)
testing for all diseases. Also, they are not much favorable for looking for such secondary findings unrelated to the disease. So we need to think Indian scientists and Indian policymakers as well as Indian lay persons who are leaders, maybe teachers, psychologists and religious leaders, they have to sit together and make guidelines and guide the society so that we don't get

Akanksha (01:03:54.446)
Thank you.

Shubha Phadke (01:04:07.091)
carried away by the sort of scientific over enthusiasm and start providing this type of testing which may be the marketing strategy of the private companies.

Akanksha (01:04:23.518)
I think that's very correctly said, ma'am. And I must say that that was an incredibly rich and insightful conversation. You have really broken down some complex ideas with such clarity and warmth for all of us. And before we close, is there a final message you'd like to leave with our listeners, especially young neurologists stepping into this interface between the genetics and newborn care?

Shubha Phadke (01:04:52.949)
very important they have to now it is the time that nobody will be able to practice without concepts of basic genetics, molecular vocabulary. we know we can train some medical geneticists, but in addition, we want to and every clinician and every neonatologist should be empowered with the genetics knowledge so that they can.

use these techniques in the best possible way for the patients and again pedigree drawing. So don't think that you know my testing and the exam sequencing is costly so we can't do much but drawing a pedigree is the cheapest and first important thing and secondly again as I said that don't order exam sequencing for everybody but consider it at a very very

early stage of your evaluation of the newborn and our simple things like malformations, dysmorphism is different, inborn errors are different and then considering that birth asphyxia is always secondary to environmental factors. So all these watertight compartments are getting broken with our experience.

Akanksha (01:06:12.064)
you.

Shubha Phadke (01:06:12.785)
And hence one has to consider that all those rare disorders which will present during neonatal period and because I see what mind knows. Once you know that cyst in the brain, sometime a few years ago we also used to feel that it is secondary to the birth asphyxia or infarction. But now we know that it can be because of many single gene disorders as well. So again.

keep on visiting OMIM, keep on reading about always differential diagnosis of genetic disorders. So not always keep them as first differential diagnosis, but depending on situation, they should be kept in the list of differential diagnosis somewhere and then appropriately exam sequencing or other testing. Trio exam sequencing always to be offered, but as you know, cost increases and

In that situation, the exome sequencing of neonate is a very important strategy. Storage of blood sample on filter paper, EDTA tube, or just a piece of umbilical cord, maybe in sterile vacutainer, stored at room temperature or stored at 4 degrees, is also very, very useful in the long run. Thank you for this opportunity. I'm sure...

There may be people are always, I see neonatologists and neurologists, are knowing, understanding, reading genetics a lot. And I think this is a very, very important part of their practice of medicine. Thank you.

Akanksha (01:07:53.044)
Absolutely, ma'am. And thank you once again. It's been such a joy and honor to have you with us today. And to our listeners, thank you for tuning in. Let's keep using the power of genetics as we discussed today with responsibility, empathy, and clarity, and then keep nurturing the tiny lives with both science and our heart. So until next time, this is Incubator India signing off. Thank you.

Shubha Phadke (01:07:59.733)
you

Shubha Phadke (01:08:20.362)
Thank you.