Heterozygous IVS 1.5(G-C) & -136 (C-G) Beta globin gene detected

Dear Andy,

I have some clarifications regards to my sons reports where we had tested his samples in two places:
1) HPLC report says that he is a "Homozygous Beta Thalassemia / Beta Thalassemia major suggest.
2) Another report DNA Mutation Analysis report says that he is a "Heterozygous IVS 1.5 (G-C) Mutation in beta globin gene detected.

There is a conflict in both the reports, the samples were tested before transfusion because his hb drop down to 4.2 g/dl at his 10th month of age.
HPLC DATA :  1) HbF - 69.1%     2) HbAo - 24.2%    3) HbA2 - 4.8%

Another HPLC had tested from peripheral blood in Gulf and the results are same as above : Heterozyougs IVS 1.5(G-C) in beta globin gene detected.

In India : based on the HPLC report they told that he is a major and tranfusion has been give on two times :
one time to rise his Hb from 4.2 to higher.
another time to maintain his Hb from above 9 to 12 which is after 18 days and now 60 days finish and we haven't transfuse him and his present Hb is 8.8%.

My son treatment is started here in Gulf recently where I am working, I had consult to an Expert doctor and we feel that we reached to a safe and right place.

I want to have a few comments on this issue and his management and conclusion.

Warm regards,
Sys

Hi Sys,

The report says heterozygous IVS-I-5 (G->C). Is there a second mutation noted? If both genes are IVS-I-5 (G->C), it would be homozygous, but heterozygous infers two non-matching gene mutations. IVS-I-5 (G->C) is the most common mutation found in India, and when one has two genes like this, thal major occurs. Major also occurs when IVS-I-5 (G->C) is combined with many of the other mutations.

The presence of 24% HbA is confusing though, as majors normally have a much lower HbA level if they have any at all. This level looks more like thal intermedia but his early transfusion requirements would more likely place him as a major. An intermedia would not have an Hb under 6. However, if illness had been present, it could lower the Hb of an intermedia to the point where transfusions are necessary.

Where are you now located? Some of the Gulf nations have very good thal treatment, and have well trained doctors. What do the current doctors think? His Hb has held above 8 for two months, which again indicates intermedia. I would find it hard to believe the child is homozygous for IVS-I-5 (G->C). It may be that it is combined with a milder mutation that is allowing for some hemoglobin production. How old is he now?

Dear Andy,

Thank you very much for your immediate reply....!!!

I would like to correct you once again : HPLC report is saying "Homozygous Beta Thalassemia" and
DNA Mutation is saying "Heterozygous for IVS 1.5 (G-C) Mutation in beta globin gene sequencing.

My son is now started 14th month, still I could not able to conclude him at this point of time.

My treating doctor is very professional and he wants to repeat HPLC once again and would like to observe him for one more month with a keen check-up on every 10-12 days without any stress on the bore marrow, this may be a touch decision but otherwise we never know he is a Intermedia / Major?

Please tell me some more details about the second mutation, which is required to be check from the patient or from parents blood sample?

Warm regards,
Sys

Sys,

Heterozygous means that the two genes are not the same gene. When a DNA test says homozygous it means that the two genes are the same. (Homozygous beta thal is a general description and does not refer to the genetic report). Knowing which two genes are involved can help with the long term prognosis, but the information you have provided only refers to the one gene. Both genes would normally be reported in the DNA testing. If both genes were the IVS-I-5 (G->C) gene, then a severe thalassemia with an HbA of 5-8% could be expected. Your child has shown a much higher percentage of HbA, but at his age this can still change and I assume this is one reason the doctor wants to repeat the HPLC testing. At 14 months, this report should be more definitive because the changes that occur after birth should be completed by now. If the HbA is still higher than 10% it may be assumed that the second mutation is of a milder nature and the gap between transfusions may be longer. It may also mean that the child may be a candidate for hydroxyurea therapy, but this will not be clear until further testing is done. If the HbA has dropped significantly, it would be a sign that regular transfusions will most likely be required.

Dear Andy,

Thanks for your prompt replies and detail updates.

In response to your reply earlie : Is about Second Mutation noted ? what is the name of this second mutation and how this has to be done, since we are planning to repeat my son's HPLC, the same time I can go for the second mutation ?

Do you think parents DNA mutation will be helpful to diagnose the case in detail, if so, what is the name of that that DNA Mutation analysis ?

Kindly do reply to me at the earliest please.

With regards,
sys

Sys,

The report you provided only mentions the one gene and it says heterozygous, which means the two beta genes can't be identical.

DNA Mutation is saying "Heterozygous for IVS 1.5 (G-C) Mutation in beta globin gene sequencing.

Was there any other gene mentioned in your report? Perhaps mention of a codon?

Dear Andy,

You are really a great...Thanks for immediate reply of my queries....!!!

The DNA report was having some limitations while we did the test for my son as they told me that second mutation not got ?

Limitations :
Only the coding regions of the beta globin gene and immediate flanking intron sequences were examined.  Changes in the promoter region, farther into the introns, or in the other non-coding regions of the gene would not be detected.  The sensitivity of the DNA sequencing is over 99% for the detection of nucleotide base changes, small deletions and insertions in the regions analyzed.  Mutations in gene other than beta globin gene would not be identified.  Miltiple exon deletions, multiple exon insertions, and complete deletion of one allele may not be identified using these methods.

You can better understand the above limitations and explain to me, and further more please let me know if as a parents we want to do our DNA test with regards to diagnose my son's case, then what is the name of that DNA test?
If we want to see other side mutation of my son then what is the name of that DNA test ? I hope you understand my query ?

With regards,
Sys

Sys,

Heterozygous means that the two genes are not the same gene. When a DNA test says homozygous it means that the two genes are the same. (Homozygous beta thal is a general description and does not refer to the genetic report). Knowing which two genes are involved can help with the long term prognosis, but the information you have provided only refers to the one gene. Both genes would normally be reported in the DNA testing. If both genes were the IVS-I-5 (G->C) gene, then a severe thalassemia with an HbA of 5-8% could be expected. Your child has shown a much higher percentage of HbA, but at his age this can still change and I assume this is one reason the doctor wants to repeat the HPLC testing. At 14 months, this report should be more definitive because the changes that occur after birth should be completed by now. If the HbA is still higher than 10% it may be assumed that the second mutation is of a milder nature and the gap between transfusions may be longer. It may also mean that the child may be a candidate for hydroxyurea therapy, but this will not be clear until further testing is done. If the HbA has dropped significantly, it would be a sign that regular transfusions will most likely be required.

Hi,
Dear Andy,

Please required urgent help on the above comments which you had reply to me couple of months before.

I had repeat my son's HPLC after 85 days from the transfusion and his results are reveals very different:
1)HbF- 11.2%  2) HbA2 - 3.6%  3) HbA - 82.7%.

His previous HPLC - HbA - 24.2% at the time when he was first diagnosed and his HB was 4.2% g/dl.

I had given transfusion, when he falls at 5.9% which is after 85 days, and another time he falls at 6.3% which is after 15 days.

Kindly advise me urgently to help me in this matter, my son case is very confusing me. Do we need to check his Alpha status?

With regards,
Sys

Hi Sys,

I can't imagine that alpha thal is also involved as he HbF level would not be high. The long interval between transfusions continues to suggest thal intermedia, but transfusion dependent intermedia. The electrophoresis is affected by the transfused blood, so it is not as useful a tool at this point. I think to ensure proper growth and development, a regular transfusion schedule will need to be implemented. Ideally, the Hb should not drop below 9. What has the doctor suggested as far as regular transfusion?

Hi Sys,

I can't imagine that alpha thal is also involved as he HbF level would not be high. The long interval between transfusions continues to suggest thal intermedia, but transfusion dependent intermedia. The electrophoresis is affected by the transfused blood, so it is not as useful a tool at this point. I think to ensure proper growth and development, a regular transfusion schedule will need to be implemented. Ideally, the Hb should not drop below 9. What has the doctor suggested as far as regular transfusion?

Dear Andy,
Thanks for your quick reply of my query.

Doctor wants to maintain him from 6~8 and he is not treating as Thalassemia since his HbA is now more than 82% and wants to investigate more about alpha gene ? Requesting to do Parents HPLC , CBC and REtuculocyte for more study. Maintaining from 6~8 will be harm in the future ? His growth looks bit OK, Kindly advise....??

With regards,
Sys

Dear Andy,
Thanks for your quick reply of my query.

Doctor wants to maintain him from 6~8 and he is not treating as Thalassemia since his HbA is now more than 82% and wants to investigate more about alpha gene ? Requesting to do Parents HPLC , CBC and REtuculocyte for more study. Maintaining from 6~8 will be harm in the future ? His growth looks bit OK, Kindly advise....??

With regards,
Sys

Hi,
Dear Andy,
Kindly give some feedback about my queries.....Also you are telling me that ....HPLC will not be helpful at this point?
According to the doctor..the HPLC value - HbA should be around 30-35% due to transfused blood but there is a tremendous change in the HbA of 82.7% which is quite confusing.  How you identified that Alpha also involved?
Please reply to me at the earliest.

With regards,
Sys

Electrophoresis is not accurate until 2-3 months after the last transfusion, so the value of the test is dependent on how long it was since the most recent transfusion. The test after 85 days should give a clear picture but the tests are completely different from the previous test. Is it possible for you to either post or send as a PM his complete report from the DNA test that concluded

"Heterozygous for IVS 1.5 (G-C) Mutation in beta globin gene sequencing

? This refers to only one of the two beta globin genes and identification of the other gene would help to clear things up.

I did not identify that alpha is involved. I said that it seems most unlikely that it could be involved because the level of HbF and HbA2 are too high to suspect alpha thal.

Electrophoresis is not accurate until 2-3 months after the last transfusion, so the value of the test is dependent on how long it was since the most recent transfusion. The test after 85 days should give a clear picture but the tests are completely different from the previous test. Is it possible for you to either post or send as a PM his complete report from the DNA test that concluded ? This refers to only one of the two beta globin genes and identification of the other gene would help to clear things up.

I did not identify that alpha is involved. I said that it seems most unlikely that it could be involved because the level of HbF and HbA2 are too high to suspect alpha thal.

Dear Andy,
Thank you for your continuous support and prompt replies for my queries.

Please find attached herewith the DNA report and the HPLC report.

Expecting your comments upon reviewing of these reports.

With regards,
Sys

The electrophoresis from the July 30 report indicates HbF, HbA and HbA2 levels that would indicate thalassemia intermedia. These levels are now completely different, with a much closer to normal HbA. The DNA report does only identify the one mutation. By itself, this cannot cause thal major. I suspect a second silent beta mutation. These are not as easily identified, but an expert may find that there is a small amount of a variant hemoglobin present. This will sometimes be found to be 2-3% of the total hemoglobin. Your current doctor may suspect this and I would talk to him in advance of HPLC testing to see if the test can find these variant hemoglobins that are not normally screened for. If a silent mutation is present, thal intermedia may be the correct diagnosis.

I have a question. Have both parents been tested as carriers or is it assumed that both are?

The electrophoresis from the July 30 report indicates HbF, HbA and HbA2 levels that would indicate thalassemia intermedia. These levels are now completely different, with a much closer to normal HbA. The DNA report does only identify the one mutation. By itself, this cannot cause thal major. I suspect a second silent beta mutation. These are not as easily identified, but an expert may find that there is a small amount of a variant hemoglobin present. This will sometimes be found to be 2-3% of the total hemoglobin. Your current doctor may suspect this and I would talk to him in advance of HPLC testing to see if the test can find these variant hemoglobins that are not normally screened for. If a silent mutation is present, thal intermedia may be the correct diagnosis.

I have a question. Have both parents been tested as carriers or is it assumed that both are?

Dear Andy,

Thanks for your valuable comments :

It has been confirmed that we both are Thalassemia Trait or Thalassemia Carriers

My HPLC values are as follows :
1)HbF - 0.2      2) HbA2 - 4.4   3) HbA - 86.5

My Wife HPLC values are as follows :
1)HbF - 0.5      2) Hbs - 0.5     3) HbA2 - 4.8     4) HbA - 84.4

I am giong to do DNA studies for my family and mine.

How do we find the silent mutation based on which type of test we need to do ?

With regards to my son thalassemia management, we are not giving him regular transfusion.
Following doctors advise  maintaining him from 6% ~ 8%, is it will be OK or we have to keep him above 9 ? Kindly advise please.

Thanks you once again.

With regards,
Sys