Of Chinese T1Dclin Patients, 27% Were Antibody Negative
By radioimmunoassay, 53.7%, 40.7%, and 33.8% of the patients were positive, respectively, for GADA, ZnT8A, and IA-2A. Negativity for all three radioimmunoassays was 27.4% (Supplementary Fig. 1).
Monogenic Diabetes in 22% of Chinese Autoantibody-Negative T1Dclin Patients
We had genomic DNA suitable for WES from 82 of these autoantibody-negative patients. Among these 82 exomes, the yield of synonymous variants per patient per gene was not significantly different from that of the 866 Chinese control subjects (mean ± SEM 0.72 ± 0.26 vs. 0.82 ± 0.32, respectively; P = 0.81). Principal components analysis, based on the exome variants, showed complete overlap of the two population samples (Supplementary Fig. 2). Of our 82 case subjects, 18 (22.0%) (95% CI 14.6–32.0) had variants likely to represent disease-causing mutations (Table 1)—a percentage several-fold higher than that reported with European ancestry cohorts defined by similar, though not identical, criteria (7,14). All were predicted to be pathogenic by three or more of the five algorithms. Variants in six patients met ACMG-AMP (13) criteria for very strong (PVS1) and another four for strong (PS3 or PS4) evidence of pathogenicity. All 18 had, at the very least, moderate pathogenicity evidence (PM2). It is important to note that these ratings rely on features other than computational prediction of pathogenicity, and, therefore, they constitute completely independent evidence. Three additional patients, not counted here, narrowly missed our predetermined MAF cutoff (Supplementary Table 3) but also likely have monogenic diabetes.
The most common gene mutated was HNF1A (maturity-onset diabetes of the young [MODY3]) with 6 of 18 patients. Two mutations were null, and one was absent from all databases. All were either truncating or rated deleterious/disease causing by four or all five of the five algorithms (Table 1). Some were previously reported with MODY3 (Supplementary Table 4). Among the 866 control subjects, there were only 2 patient with HNF1A variants meeting the same predetermined criteria. Thus, Pobs = 7.3% (95% CI 3.9–14.4) of autoantibody-negative T1Dclin Chinese patients have HNF1A mutations. Pexp, derived from the control subjects, was 0.2% (95% CI 0.08–0.4%) with P = 6.5 × 10−6, false discovery q = 0.03.
Somewhat surprisingly, almost as common as MODY3 were diallelic variants of WFS1, recessively mutated in WS, in four case subjects not reported to have any other syndromic features and initially recruited for the T1D GWAS. One was homozygous, and in another two cases, the two variants were close enough to be confirmed in trans by alignment inspection (Supplementary Fig. 3). All of these variants were rated deleterious by at least four of the five algorithms, and two have previously been reported in cases of fully expressed WS (Supplementary Table 4). Only one of the 866 control subjects had two missense mutations, both predicted benign by all five algorithms. For 4 of 82 vs. 0 of 866, Pobs = 4.9% (95% CI 2.4–11.1) vs. Pexp = 0% (95% CI 0–0.6), P = 6.5 × 10−6, false discovery q = 0. Thus, by our best estimate, 5% of autoantibody-negative Chinese T1Dclin case subjects have nonsyndromic diabetes due to WFS1 mutations.
One patient was thoroughly examined and found to be normal by fundoscopic eye exam, audiogram, and urine density (Supplementary Fig. 4), confirming the reported absence of other WS manifestations (optic nerve atrophy, hearing loss, and diabetes insipidus) in that case.
The remaining six patients had mutations in other MODY genes (Table 1), but the numbers were too small for statistics (Supplementary Table 2). Two patients had mutations in KLF11 (MODY7) and PAX4 (MODY9), OMIM (Online Mendelian Inheritance in Man) genes whose role in monogenic diabetes remains unconfirmed. Only one KLF11 mutation met pathogenicity-prediction criteria, versus two control subjects (P = 0.25), not providing support for this gene. NEUROD1 is better established as the cause of MODY6 (15) and supported by our finding of a mutation in one patient (deleterious by all 5 algorithms) vs. 0 of 866 control subjects. Other well-established genes found to be mutated were HNF1B, ABCC8, and GCK (each in two patients) and INS (one patient).
Both HNF1B mutations were complete gene deletions within the known recurrent microdeletion at 17q22, reported to account for as many as 50% of MODY5 cases (16). The two patients had LOH over at least 1.4 Mb that encompassed HNF1B among 28 genes (Fig. 1, top). Over the LOH region in each patient, each of 178 exons had approximately half the read counts of the average of all other patients (Fig. 1, bottom); P = 7.5 × 10−15 and 1 × 10−16 for patients 17 and 18, respectively. Adjacent to the deletion, there were no shared haplotypes, indicating independent occurrence in two different ancestral chromosomes.
Fasting C-peptide, available in 56 of 82 patients, varied widely and was no different between patients with or without a mutation (mean 249.5 vs. 280.5, P = 0.6699). (Supplementary Fig. 5A).
For the 82 patients (37 females), the median age at diagnosis was 20 years (range 1–61). Patients with mutations were younger: median age at diagnosis 13.5 vs. 23.2 years (P = 0.0297) (Supplementary Fig. 5B).
In a parallel study, 126 T1Dclin autoantibody-negative patients, including 44 whose DNA sample was unsuitable for WES, were tested for mtDNA mutations by Sanger sequencing. Four had the m.3243A>G mutation and two the m.3316A>G, both previously reported in Chinese patients with diabetes (17,18). Heteroplasmy, estimated from the sequencing peaks, ranged from 6.2 to 50.4% (Supplementary Table 5)—well within the described range (19).
The distribution of genetic causes of 18 confirmed cases of monogenic diabetes and 6 cases of mitochondrial diabetes is shown in Supplementary Fig. 6.