GCK-MODY NIPT

Glucokinase-Maturity-Onset Diabetes of the Young (GCK-MODY) is a common genetic cause of diabetes. It causes a high fasting blood glucose from birth, but it does not normally cause any health complications. However, it is important to know about in pregnancy. This is because the baby’s birth weight is decided by whether they have inherited the mutation in the glucokinase gene (GCK) from their mother or not. The mother’s glucose crosses the placenta and when the baby has not inherited their mother’s mutation, they sense this as being high and secrete insulin. Insulin promotes growth and these babies can become very large. This has the potential to cause complications in labour and may increase their risk of low blood sugar needing special care once they are born. When the baby has inherited their mother’s mutation, they sense glucose at a similar level and secrete an appropriate amount of insulin. In these cases the baby is expected to have a birth weight within the normal range.

It is possible to treat the mother with insulin in pregnancy to try and reduce blood glucose levels and reduce the risk of large babies who have not inherited their mother’s mutation. However, this is not indicated where the baby has inherited the maternal mutation and it may increase their risk of being too small.

To try and avoid unnecessary insulin treatment, doctors try to predict whether the baby has inherited the mother’s mutation or not by looking at ultrasound scans, which usually start from 24-28 weeks of pregnancy. If the baby has a large abdominal circumference, it is assumed that they have not inherited their mother’s mutation. At present, the recommended abdominal circumference threshold is the 75^th percentile. Babies with an abdominal circumference less than this would not be treated. However, it is not known how accurate this is at correctly predicting the baby’s mutation status.

An alternative test where the baby’s DNA is analysed from the mother’s blood sample could be more useful. This test is called non-invasive prenatal testing (NIPT). Preliminary studies have shown that it is possible to get a result from a blood sample taken as early as 12 weeks of pregnancy.

To know if non-invasive prenatal testing should be recommended for all women with GCK-MODY we need to find out if it is more accurate than current practice with ultrasound and if it is feasible. To do this we plan to compare their accuracy and see how often and when a result can be given with non-invasive testing in these pregnancies.

Glucokinase-Maturity-Onset Diabetes of the Young

Heterozygous inactivating mutations in the glucokinase gene (GCK) result in a mild fasting hyperglycemia (5.5-8 mmol/L) known as GCK-Maturity-Onset Diabetes of the Young (GCK-MODY).(1) It is not normally associated with long-term complications and does not require treatment.(2) The estimated population prevalence is 0.1 in 100, increasing to 0.9 in 100 in gestational diabetes.(3) It is present from birth (4) but is frequently diagnosed in later-life, particularly in pregnancy. This is because women have features atypical for gestational diabetes, such as a normal BMI and a history of normal birth weight children despite high glucose levels in pregnancy, prompting clinicians to consider alternative diagnoses.

Implications of maternal GCK-MODY in pregnancy

Birth weight and pregnancy outcomes in GCK-MODY pregnancies are determined by fetal genotype.(5) Where the fetus has not inherited the maternal GCK mutation there is a higher risk of large-for-gestational-age (LGA, birth weight >90^th percentile for sex and gestational age) and macrosomia (birth weight > 4 kg) as a result of higher fetal-insulin mediated growth in response to maternal hyperglycaemia.(6) This is associated with a higher risk of operative delivery and potential complications.(5) Fetuses who inherit the maternal mutation sense glucose at a higher level and hence secrete less insulin, resulting in a normal birth weight and LGA rates comparable with the general population. The difference in birth weight between babies who have and have not inherited the maternal mutation is ~500-600 g.(5,6) It is possible to treat women with insulin to reduce the risk of fetal overgrowth in pregnancies where the fetus does not have GCK-MODY. However, this is not beneficial in baby’s who have inherited the maternal mutation and may be harmful as it increases the risk of small-for-gestational-age (SGA, birth weight <10^th percentile for sex and gestational age).(7)

Current approach to management of GCK-MODY pregnancies

If the fetus has inherited the maternal mutation insulin treatment is not recommended. As fetal GCK-MODY is not lethal or life-limiting, invasive testing with chorionic villous sampling or amniocentesis is not recommended due to the ~1% risk of fetal loss. At present, the circumference of the fetal abdomen is used to determine whether the fetus has inherited the maternal mutation or not. If the fetus has an abdominal circumference >75^th percentile from 24 weeks gestation, this suggests that the baby is at higher risk of LGA and therefore more likely not to have GCK-MODY.(5,8,9) This threshold was selected based on randomised control trials of ultrasound vs usual management of gestational diabetes and was found to be safe.(10,11) An abdominal circumference >90^th percentile has also been suggested as a possible threshold.(8) When there is a raised fetal abdominal circumference insulin treatment may be trialled, whereas it is not given to women where the fetus is growing normally. The accuracy of ultrasound to predict fetal genotype has not previously been determined so it is not known whether this is the optimal way to manage these pregnancies.

Non-invasive prenatal testing for fetal genotype

An alternative approach to predicting fetal genotype is to use non-invasive prenatal testing (also known as NIPT). This involves testing the cell-free DNA in maternal blood. A method for this using digital droplet PCR was validated by the Exeter Genomics Laboratory.(12) It does not have the risks of fetal loss seen with invasive testing and it was demonstrated to be highly accurate, with a sensitivity and specificity of 100% where a result was reportable.(12)

Potential impact of non-invasive prenatal testing

If non-invasive prenatal testing is more accurate than ultrasound, fewer fetuses would be misclassified and a more tailored approach to pregnancy management could be instigated. This could prevent unnecessary insulin treatment in pregnancies where the fetus has also inherited the maternal mutation. Apart from the potential risk of restricting fetal growth, many women find insulin treatment challenging, particularly in GCK-MODY (personal communications, Dr Hughes/Prof Hattersley). This is because of counter-regulation, meaning very high doses of insulin are needed and women may suffer from symptoms of hypoglycaemia.(13) The validation of the non-invasive prenatal testing method showed it to be accurate for samples taken as early as 12 weeks gestation, so it is also possible that it could provide results prior to ultrasound. This could be helpful where the fetus has not inherited the maternal mutation, allowing treatment to start near the beginning of the third trimester, potentially mitigating insulin-mediated fetal growth which starts at this time. Regardless of timing, knowledge of an unaffected fetus could be beneficial for planning postnatal management, since they are at risk of neonatal hypoglycaemia.(5)

What evidence is necessary to implement non-invasive prenatal testing on a wider scale?

For a specific test to be considered worthwhile and recommended as standard practice, it should show benefits over existing practice. This study, which specifically compares non-invasive prenatal testing with ultrasound-guided management, will obtain evidence to investigate this.

Aim

To determine the diagnostic accuracy and feasibility of non-invasive prenatal testing of fetal genotype in pregnancies where the mother has GCK-MODY.

Primary objective

To compare the diagnostic accuracy of non-invasive testing compared with ultrasound in pregnancies where the mother has GCK-MODY.

Secondary objective

To determine the feasibility of non-invasive prenatal testing in practice by investigating:

1. The proportion of pregnancies receiving a reportable result.
2. The number of samples required for a result.
3. Timing of result from receipt of a blood sample in the laboratory.

4.1 Summary of design

An analysis of a consecutive series of participants who contributed blood samples for non-invasive prenatal testing.

This is a single centre study, but samples were received from women internationally. Samples were tested by the Exeter Genetics Laboratory and stored under the remit of the Genetic Beta Cell Research Bank.

We would like to conduct an analysis of samples that have undergone non-invasive testing and obtain information on their pregnancies. Based on average enquiry rates for non-invasive testing in GCK-MODY, we aimed to collect information on 30 pregnancies where the result was given to a clinician to meet the secondary objective, but we went back to all clinicians for pregnancy information to fulfil the primary objective.

The non-invasive prenatal testing has been performed using the existing methods.(12) Referring clinicians were contacted to collect clinical information from medical records.

4.2 Eligibility Criteria

Inclusion criteria:

• A mother with GCK-MODY who has undergone non-invasive prenatal testing.

Exclusion criteria:

• Lack of consent for their samples to be used for research purposes.

4.3 Assessment and Management of Risk

This is a low risk study which categorised as ‘a study limited to working with human tissue samples and/or analysis of data’. Included women will have undergone a blood test which has potential risks and complications, but these are limited to bruising, pain and the potential for repeat blood sampling.

5.1 Sample Collection and Storage

Blood samples have been collected from women in pregnancy and at delivery from umbilical cord and stored in the Genetic Beta Cell Research bank. Where umbilical cord blood is not available a buccal swab from the child from the pregnancy will be collected post-delivery to confirm the non-invasive prenatal test result.

The samples have been stored with the appropriate consent to be used for future ethically approved research.

5.2 Sample Transfer

Samples have been transferred to the Exeter Genomics Laboratory, at the Royal Devon University Healthcare NHS Foundation Trust for analysis.

Samples transferred are anonymised by assigning a unique number/identifier with the file linking the code to personal identifiable information about the donor held securely in a database.

5.3 Sample Analysis

Genetic testing is performed using the existing methods and protocol. These analyses are performed by state registered NHS laboratory technicians using a previously published method (12) in a routine diagnostic setting. Remaining sample left after non-invasive testing are stored in the Genetic Beta Cell Research Bank.

6.1 Data Collection, Transfer and Storage

We will be collecting clinical information about the pregnancies from these samples.

The responsible clinician has or will provide the research team with anonymised routinely collected data about the appropriate blood donors including but not limited to:

• Mother’s characteristics (age, BMI, fasting glucose)
• Mother’s treatment in pregnancy
• Fetal ultrasound measurements
• Baby’s sex
• Baby’s date of Birth
• Gestational age at birth
• Birth weight
• Information on neonatal complications

This information will have been collected with the samples or retrospectively using a clinical data collection questionnaire. The clinical data collection questionnaire is sent via secure nhs.net email. Data transferred to the Exeter research team will be anonymised, each data set will be assigned a unique number/identifier matching the tissue sample and stored securely.

Once the analyses have been completed, the results will be checked by the chief investigator and transferred to a database. All donor research data will be identified by unique ID numbers and will be held on encrypted devices.

Results from the non-invasive prenatal test were provided to clinicians from 2019 as part of the genetics diagnostics service after the method was validated, sent as an official report via a secure nhs.net email.

6.2 Source Data

Data collection forms, laboratory results and will be deemed source data.

6.3 Data Confidentiality

The research team in Exeter will have access to tissue samples and data. Blood samples and data transferred from clinicians to the Exeter research team will be assigned a unique number/identifier.

6.4 Data Archiving

At the end of the study, archiving will be carried out in accordance with sponsor protocols and, where relevant, local site governance requirements.

6.5 Data Availability

The data will not be freely available to protect the interests of research participants. However, summarised data will be published and requests from researchers to access the data will be considered through managed open-collaboration.

This is an investigation to establish the diagnostic accuracy of non-invasive testing with ultrasound. We will approach up to 35 who underwent testing when the test was validated. As we are also interested in the practicalities of testing, we aim to conduct an analysis of up to 30 pregnancies where a result was issued to a clinician. This proposed sample size represents the approximate number of enquiries regarding non-invasive testing over a 24 month period.

The chief investigator will set up and organise the day-to-day operation of the study.

Disclosure of results and patient feedback

Since the non-invasive prenatal testing method has been validated, reportable results are fedback to the referring clinician. Confirmatory results from umbilical cord blood and saliva samples are also fedback. The information will be provided in a formal genetics diagnostic report and sent via secure nhs.net email.

The clinician will deal with results and will feedback to the patient.

The study will finish when all analysis of samples and data has been undertaken.

Non-invasive prenatal testing between 2019 and 2021 was funded by a Wellcome and University of Exeter funded GW4-Clinical Academic Training PhD fellowship to the chief investigator.

Monitoring of this study will ensure compliance with Good Clinical Practice.

NHS Indemnity will apply.

We hope that the results generated from this study will help inform decisions for routinely recommending non-invasive prenatal testing in pregnancies where the mother has GCK-MODY.

The chief and co-investigators have spoken to women with GCK-MODY and their experiences of their previous pregnancies. The chief and co-investigators are available to answer any questions about the work and will seek feedback from participants on their experience of testing where possible.

Results will be written up and submitted for publication in a peer-reviewed journal. Abstracts will be submitted to national and international conferences. Results will be presented to clinical colleagues at regular in-house meetings. The evidence from this study may form part of an application for funding of the test on a national basis.

1. Gloyn AL. Glucokinase (GCK) mutations in hyper- and hypoglycemia: Maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemia of infancy. Human Mutation. 2003;22(5):353–62.
2. Steele AM, Wensley KJ, Ellard S, Murphy R, Shepherd M, Colclough K, et al. Use of HbA1c in the Identification of Patients with Hyperglycaemia Caused by a Glucokinase Mutation: Observational Case Control Studies. PLoS One [Internet]. 2013 Jun 14 [cited 2020 Apr 10];8(6). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683003/
3. Chakera AJ, Spyer G, Vincent N, Ellard S, Hattersley AT, Dunne FP. The 0.1% of the Population With Glucokinase Monogenic Diabetes Can Be Recognized by Clinical Characteristics in Pregnancy: The Atlantic Diabetes in Pregnancy Cohort. Diabetes Care. 2014 May 1;37(5):1230–6.
4. Hughes AE, De Franco E, Globa E, Zelinska N, Hilgard D, Sifianou P, et al. Identification of GCK-maturity-onset diabetes of the young in cases of neonatal hyperglycemia: A case series and review of clinical features. Pediatr Diabetes. 2021 Sep;22(6):876–81.
5. Spyer G, Macleod KM, Shepherd M, Ellard S, Hattersley AT. Pregnancy outcome in patients with raised blood glucose due to a heterozygous glucokinase gene mutation. Diabetic Medicine. 2009;26(1):14–8.
6. Hattersley AT, Beards F, Ballantyne E, Appleton M, Harvey R, Ellard S. Mutations in the glucokinase gene of the fetus result in reduced birth weight. Nature Genetics. 1998 Jul;19(3):268–70.
7. Dickens LT, Letourneau LR, Sanyoura M, Greeley SAW, Philipson LH, Naylor RN. Management and pregnancy outcomes of women with GCK-MODY enrolled in the US Monogenic Diabetes Registry. Acta Diabetol. 2019 Apr;56(4):405–11.
8. Chakera AJ, Carleton VL, Shields B, Ross GP, Hattersley AT. Response to Comment on: Chakera et al. Antenatal diagnosis of fetal genotype determines if maternal hyperglycemia due to a glucokinase mutation requires treatment. Diabetes Care 2012;35:1832-1834. Diabetes Care. 2013 Jan;36(1):e15.
9. Spyer G, Hattersley AT, Sykes JE, Sturley RH, MacLeod KM. Influence of maternal and fetal glucokinase mutations in gestational diabetes. Am J Obstet Gynecol. 2001 Jul;185(1):240–1.
10. Buchanan TA, Kjos SL, Montoro MN, Wu PY, Madrilejo NG, Gonzalez M, et al. Use of fetal ultrasound to select metabolic therapy for pregnancies complicated by mild gestational diabetes. Diabetes Care. 1994 Apr;17(4):275–83.
11. Schaefer-Graf UM, Kjos SL, Fauzan OH, Bühling KJ, Siebert G, Bührer C, et al. A Randomized Trial Evaluating a Predominately Fetal Growth–Based Strategy to Guide Management of Gestational Diabetes in Caucasian Women. Diabetes Care. 2004 Feb 1;27(2):297–302.
12. Caswell RC, Snowsill T, Houghton JAL, Chakera AJ, Shepherd MH, Laver TW, et al. Noninvasive Fetal Genotyping by Droplet Digital PCR to Identify Maternally Inherited Monogenic Diabetes Variants. Clin Chem. 2020 Jul 1;66(7):958–65.
13. Chakera AJ, Steele AM, Gloyn AL, Shepherd MH, Shields B, Ellard S, et al. Recognition and Management of Individuals With Hyperglycemia Because of a Heterozygous Glucokinase Mutation. Diabetes Care. 2015 Jul;38(7):1383–92.