A biomarker-based screening method assessing levels of C-peptide and islet autoantibodies in patients with diabetes is an effective, inexpensive approach
to identify patients with monogenic forms of the disease, including maturity-onset diabetes of the young, according to findings from a population-based assessment conducted in Britain.
“Identifying patients with monogenic diabetes, particularly [maturity-onset diabetes of the young], can be challenging,” Beverley M. Shields, PhD,senior lecturer in medical statistics with the Institute of Biomedical and Clinical Science at the University of Exeter Medical School, United Kingdom, and colleagues wrote.
“Monogenic diabetes is confirmed by molecular genetic testing, but this is expensive, so testing all patients is not feasible. An approach that could be used to enrich for monogenic diabetes, increasing the proportion identified in those who undergo genetic testing, would be helpful.”
Shields and colleagues tested a screening pathway using both C-peptide (via urinary C-peptide to creatinine ratio) and glutamic acid decarboxylase (GAD) and insulinoma-associated-2 autoantibodies (IA-2A) to exclude type 1 diabetes in two populations with previously high pickup rates of maturity-onset diabetes of the young (MODY) — patients diagnosed before age 30 years and currently younger than 50 years from the areas surrounding Royal Devon and Exeter NHS Foundation Trust (n = 716) and Ninewells Hospital (n = 702), both in the United Kingdom. For all patients negative for antibodies with significant endogenous insulin, DNA sequencing was performed for known MODY-related mutations.
Within the cohort, 1,365 had no known genetic cause for their diabetes, 34 had confirmed monogenic diabetes at baseline and eight had cystic fibrosis-related diabetes. After urinary C-peptide to creatinine testing, 979 (76%) had minimal endogenous insulin secretion, indicating type 1 diabetes, and received no further testing. Of the 386 patients then tested for GAD or IA-2A autoantibodies, 170 (44%) tested positive, also indicating type 1 diabetes, and received no further testing.
The remaining 216 patients underwent sequencing for the three most common MODY-related genes; eight tested positive, according to researchers. Of the 208 who tested negative for common MODY genes, additional testing by targeted, next-generation sequencing identified mutations in genes associated with monogenic diabetes in eight more patients. One additional patient had a MODY-related mutation identified through exome sequencing. The results suggested a prevalence of 3.6% (95% CI, 2.7-4.7) among the 1,407 recruited participants.
“A total of 199 out of 1,348 (15%) patients were put forward for genetic testing who were not found to have monogenic diabetes (ie, 15% false-positive rate, so 85% specificity),” the researchers wrote. “Assuming a 98% sensitivity and 85% specificity, the [positive predictive value] for the pathway is 20%, suggesting a 1-in-5 pickup rate for monogenic diabetes, a 5.6-fold increase in probability over the background prevalence alone.”
The strength of the pathway, the researchers wrote, is in the integration of both C-peptide and islet autoantibodies, rather than relying on clinical features.
“This offers a simple approach that does not require specific clinician interpretation or complex algorithms of different combinations of features,” the researchers wrote. “By combining the two biomarkers, we increase the discriminatory ability and allow the clinician to pick up even atypical cases and rarer forms of monogenic diabetes, which traditional criteria may miss. The use of clinical features, however, results in fewer cases being sent for genetic testing that are negative, which clearly has cost implications.”
The most cost-effective approach will likely involve a combination of both biomarkers and clinical features, they noted, and further research is needed to determine whether the pickup rate could be improved by integrating the pathway with clinical features, such as the MODY calculator. 
Disclosures: The authors report no relevant financial disclosures.
Population-Based Assessment of a Biomarker-Based Screening Pathway to Aid Diagnosis of Monogenic Diabetes in Young-Onset Patients
Beverley M. Shields,1,2
Maggie Shepherd,1,2 Michelle Hudson,1
Timothy J. McDonald,1,3 Kevin Colclough,4
Jaime Peters,5 Bridget Knight,1,2
Chris Hyde,5 Sian Ellard,1,4
Ewan R. Pearson,6 and
Andrew T. Hattersley,1,2 on behalf of the
UNITED study team
Monogenicdiabetes,ayoung-onsetformofdiabetes,isoftenmisdiagnosedastype1 diabetes, resulting in unnecessary treatment with insulin. A screening approach for monogenic diabetes is needed to accurately select suitable patients for expensive diagnostic genetic testing. We used C-peptide and islet autoantibodies, highly sen-sitive and specific biomarkers for discriminating type 1 from non–type 1 diabetes, in a biomarker screening pathway for monogenic diabetes.
We studied patients diagnosed aged 30 years or younger, currently younger than
50 years,intwoU.K. regions with existinghighdetectionofmonogenicdiabetes. The biomarker screening pathway comprised three stages:1) assessment of endogenous insulin secretion using urinary C-peptide/creatinine ratio (UCPCR); 2) if UCPCR was ‡0.2 nmol/mmol, measurement of GAD and IA2 islet autoantibodies; and 3) if negative for both autoantibodies, molecular genetic diagnostic testing for 35 mono-genic diabetes subtypes.
A total of 1,407 patients participated (1,365 with no known genetic cause, 34 with monogenicdiabetes,and8withcystic fibrosis–relateddiabetes).Atotalof386outof 1,365 (28%) patients had a UCPCR ‡0.2 nmol/mmol, and 216 out of 386 (56%) were negative for GAD and IA2 and underwent molecular genetic testing. Seventeen new casesofmonogenicdiabetes werediagnosed(8commonMaturityOnset Diabetesof the Young [Sanger sequencing] and 9 rarer causes [next-generation sequencing]) in addition to the 34 known cases (estimated prevalence of 3.6% [51/1,407] [95%CI 2.7–4.7%]). The positive predictive value was 20%, suggestinga one-in-five detection rate for the pathway. The negative predictive value was 99.9%.
Thebiomarker screeningpathwayfor monogenic diabetes isaneffective,cheap,and easily implemented approach to systematically screening all young-onset patients. The minimum prevalence of monogenic diabetes is 3.6% of patients diagnosed aged 30 years or younger.
From the article
Correct classification of a patient’s diabe-tes is important to ensure he or she re-ceives the most appropriate treatment and ongoing management. The most common form of diabetes in children and young adults is type 1 diabetes, ac-counting for .90% of cases (1,2). Other forms of diabetes in this age group, such as monogenic diabetes (including Matu-rity Onset Diabetes of the Young [MODY]), or young-onset type 2, are not often con-sidered. It is estimated that at least 80% of patients with MODY are misdiagnosed (3), and other rarer forms of monogenic diabetes often go unrecognized because of lack of awareness (4). Patients with MODY or type 2 diabetes misclassified as type 1 diabetes will be treated with insulin, whereas noninsulin therapy would be more appropriate. Diet and metformin are the treatment of choice in young type 2 diabetes (5). Patients with MODY because of mutations in the HNF1A or HNF4A genes respond well to low-dose sulphonylureas (6,7), and those with MODY because of mutations in the GCK gene require no pharmacological treatment (8). Getting a correct diagnosis for all forms of monogenic diabetes has important implications for management of an individual’s diabetes, a prognosis, and recognitionofassociatedclinicalfeatures; it also allows appropriate counseling of other family members regarding likely inheritance (4).
Identifyingpatientswithmonogenicdi-abetes, particularly MODY, can be chal-lenging. Monogenic diabetes is confirmed by molecular genetic testing, but this is expensive, so testing all patients is not feasible. An approach that could be used to enrich for monogenic diabetes, increasing the proportion identified in those who undergo genetic testing, would be helpful. Clinical features can aid identification of those who may have an alternative diagnosis, and a prob-ability calculator has been developed to help determine which patients are likely to have the most common forms of
MODY (9). However, this will not pick up other forms of monogenic diabetes, and its performance is weaker for detecting MODY in insulin-treated patients com-pared with non–insulin-treated patients.
An alternative approach to enrich for monogenic diabetes is to use biomarkers that have been shown to discriminate well between type 1 and other forms of young-onset diabetes. Type 1 diabetes is
characterized by autoimmune destruc-tion oftheb-cells inthe pancreas, leading toabsoluteinsulindeficiency,sotwotests that could be used to diagnose type 1 di-abetes are islet autoantibodies (markers oftheautoimmuneprocess)andC-peptide (a marker of insulin deficiency). C-peptide hasbeenshowntobeahighlysensitiveand specific biomarker for discriminating be-tween type 1 and type 2 diabetes and MODY 3–5 years after diagnosis (10,11).
Urine C-peptide/creatinine ratio (UCPCR) canbeusedtoremovetheneedforblood samples, which may be of particular con-cern in the pediatric population, and means that the sample can easily be taken at home and posted to the labora-tory (12). GAD and IA2 islet autoanti-bodies also discriminate well between type 1 and MODY, with cross-sectional studies showing they are present in 80% of patients with type 1 diabetes and in ,1% of patients with MODY (13).
These biomarkers have been used to screen for MODY in other studies (14,15), but have been limited to pediatric cases only. Given the median age at diagnosis for MODY is 20 years (from U.K. refer-rals data [3]), and there is on average a delay of 13 years from diabetes diag-nosis to a confirmed genetic diagnosis (16), it is crucial to study adults as well.
Furthermore, the combined diagnostic performance of the two biomarkers as a screening pathway has not been formally assessed.
Byexcludingthosewithtype1diabetes using these two biomarkers, we can obtain a smaller percentage of patients in whom diagnostic molecular testing for monogenic diabetes could be per-formed. We tested a screening pathway using both C-peptide and islet autoanti-bodies to exclude type 1 diabetes in two populations with previously high pickup rates of MODY (3) and performed genetic testing on all patients with significant en-dogenous insulin and absence of islet autoantibodies. This allowed us to deter-mine the prevalence of all monogenic di-abetes subtypes in those diagnosed at 30 years or younger and to calculate the positive predictive values (PPVs) and neg-ative predictive values (NPVs) for the pathway.
The biomarker screening pathway for monogenic diabetes is a systematic, cheap (U.K. UCPCR cost of £10.80 and antibodies cost of £20), and easily imple-mented approach to screening all pa-tients with young-onset diabetes in a clinic or population that helps identify suitable patients for molecular diagnostic genetic testing. The pathway picked up new cases of monogenic diabetes, even in areas of existing high detection be-cause of research interests in the regions. We found 3.6% of patients diagnosed at younger than 30 years of age have
monogenic diabetes. In areas in which no cases have been identified, we estimate that 1 in 5 patients referred for genetic testing because of the pathway will have monogenic diabetes, which is a 5.6-fold higher detection rate than if all patients in this age range received genetic testing. The high NPV of 99.9% indicates it is an extremely effective approach for ruling out monogenic diabetes.
There have been relatively few studies that have systematically screened whole populations for monogenic diabetes. The majority of studies have been in pediatric populations only (14,15,22–26), with only two studies that have screened adults (27,28).Noother studyhas systematically screened a whole population of both adults and children together. Only 8 out of 51 (16%) of patients with a genetic diagnosis of monogenic diabetes in our cohort were in the pediatric age range (younger than 20 years) at the time of recruitment, highlighting the importance of looking for monogenic diabetes in adult diabetes clinics. This may explain why the prevalence we find is higher thananyofthepreviouspediatricstudies. The strength of our pathway is the in-tegration of two biomarkers (C-peptide and islet autoantibodies [both GAD and IA2]), rather than relying on clinical fea-tures. This offers a simple approach that does not require specific clinician inter-pretation or complex algorithms of different combinations of features.Weshowed that by using clinical features alone, over half of the cases of monogenic diabetes would be missed.
By combining the two biomarkers, we increase the discrimina-tory ability and allow the clinician to pick up even atypical cases and rarer forms of monogenic diabetes, which tra-ditional criteria may miss. The use of clin-ical features, however, results in fewer cases being sent for genetic testing that are negative, which clearly has cost impli-cations.Themostcost-effectiveapproach is likely to involve a combination of bio-markers and clinical features. Further studiesareneeded to determinewhether thepickupratecouldbefurtherimproved by integrating the pathway with clini-cal features, such as the MODY calcula-tor, or whether this would result in moremissedpatientsbecause ofreduced testing.
In this study, we also systematically tested all known genes for monogenic diabetes, rather than just the most com-mon MODY genes (GCK, HNF1A, and HNF4A).Nineout of17 (53%) of the cases identified as part of our cohort had mu-tations identified through additional testing on the targetedcapture,and17outof 51 (33%) of all of the monogenic diabetes cases found in total had mutations in other genes, highlighting the advantage of further testing using targeted next-generation sequencing.
Health economic evaluation of the pathwayfordetectingthecommonforms of MODY (GCK, HNF1A, and HNF4A) has been carried out as a separate project, which has shown the pathway to be cost-saving(20,21).Thecost-effectiveness of additional testing for other forms of monogenic diabetes has not been as-sessed. Because of the rarity of other monogenic diabetes, there are few data available to inform such analyses. Treat-ment change from insulin to sulphonylur-easisstillpossibleincasesdiagnosedwith ABCC8 and KCNJ11 (29,30), and for other
genes for which treatment change is not an option, a confirmed diagnosis can still help with management, prognosis, and advice on risk to other family members (4). The decision whether to pay for the more expensive, but more comprehen-sive, next-generation sequencing, rather than Sanger sequencing for MODY genes only, would depend on assessing the tradeoffs of additional costs with long-term benefits to the patient. The pres-ence of additional clinical features (e.g., renal cysts associated with HNF1B) may also point to specific monogenic diagno-ses and increase the likelihood of a posi-tive genetic test result.
A limitation of our study was that we had small numbers of patients with monogenicdiabetesonwhichto evaluate the sensitivity of the pathway. Consider-ably larger studies have shown the biomarkers individually to be highly sen-sitive for monogenic diabetes (99% for UCPCR [10,11] and .99% for islet auto-antibodies [13]), and by using both of these markers in a pathway, the number of missed cases should be minimal at a population level (2% of 3.6% = 0.07%, reflected in the NPV of 99.9%). Although there have been reports of MODY pa-tients who are positive for islet autoanti-bodies (reviewed in Ref. 13), these are rare and likely to be cases with coinciden-tal type 1 diabetes. Previous studies re-porting high prevalence of positive autoantibodies in their cohort have in-cluded clinically defined, rather than ge-netically confirmed, MODY (31) or use low cutoffs for antibody positivity, which can be inappropriate (32), and are likely to represent an overestimate. There is also the potential for missed cases based onUCPCR,butagain,thenumberofthese patients will be small, and as they have insulin levels suggestive of type 1 diabe-tes (33), they are unlikely to be able to transfer off insulin even if a genetic diag-nosis is made.
A further limitation is that despite screening using C-peptide and antibody testing, the PPV is still fairly low at 20%, indicating four out of five screened will not have a monogenic cause identified on diagnostic molecular genetic testing. However, the aim of our screening path-way is that it is used purely as a tool to narrow down those individuals who would be more appropriate for genetic testing. This approach is a vast improve-ment over no screening  represent a PPV at the background prev-alence rate of 3.6%), misses fewer cases than using clinical features alone, and is at a level that has been shown to be cost-effective (20,21). Furthermore, the screening pathway still provides useful test results for this age group that of-fer additional information to support pa-tient care. Patients with severe insulin deficiency, as determined by very low C-peptidevalues,willnotrespondtonon-insulin therapy (33). Positive C-peptide and negative antibody results are impor-tantclinicallytohighlightatypical casesof type1diabetesorinwhichother forms of diabetes, such as young-onset type 2 di-abetes, should be considered. Patients with very high endogenous insulin with-outisletautoantibodiesandnomutations in monogenic diabetes genes are likely to have type 2 diabetes and may be able to manage on noninsulin treatment.
Finally, this study comprised a 98% white population and assesses patients at a median of 14 years after diagnosis. Assessment of the pathway in other racial groups and in patients close to diagnosis is needed.
In conclusion, we have demonstrated a simple, cheap, effective screening path-way that could be implemented at a pop-ulation level to help correctly diagnose patients with monogenic diabetes.
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