The Changing Landscape of Neonatal Diabetes Mellitus in Italy Between 2003 and 2022

Context: In the last decade the Sanger method of DNA sequencing has been replaced by next-generation sequencing (NGS). NGS is valuable in conditions characterized by high genetic heterogeneity such as neonatal diabetes mellitus (NDM). Objective: To compare results of genetic analysis of patients with NDM and congenital severe insulin resistance (c.SIR) identiﬁed in Italy in 2003-2012 (Sanger) vs 2013-2022 (NGS). Methods: We reviewed clinical and genetic records of 104 cases with diabetes onset before 6 months of age (NDM + c.SIR) of the Italian dataset. Results: Fifty-ﬁve patients (50 NDM + 5 c.SIR) were identiﬁed during 2003-2012 and 49 (46 NDM + 3 c.SIR) in 2013-2022. Twenty-year incidence was 1:103 340 (NDM) and 1:1 240 082 (c.SIR) live births. Frequent NDM/c.SIR genetic defects ( KCNJ11 , INS , ABCC8 , 6q24, INSR ) were detected in 41 and 34 probands during 2003-2012 and 2013-2022, respectively. We identiﬁed a pathogenic variant in rare genes in a single proband ( GATA4 ) (1/42 or 2.4%) during 2003-2012 and in 8 infants ( RFX6 , PDX1 , GATA6 , HNF1B , FOXP3 , IL2RA , LRBA , BSCL2 ) during 2013-2022 (8/42 or 19%, P = .034 vs 2003-2012). Notably, among rare genes 5 were recessive. Swift and accurate genetic diagnosis led to appropriate treatment: patients with autoimmune NDM ( FOXP3 , IL2RA , LRBA ) were subjected to bone marrow transplant; patients with pancreas agenesis/hypoplasia ( RFX6 , PDX1 ) were supplemented with pancreatic enzymes, and the individual with lipodystrophy caused by BSCL2 was started on metreleptin. Conclusion: NGS substantially improved diagnosis and precision therapy of monogenic forms of neonatal diabetes and c.SIR in Italy.

Neonatal diabetes mellitus (NDM) is a subtype of monogenic diabetes with onset within 6 months of age (1).NDM may be isolated or syndromic, depending on the single gene involved and its mode of inheritance can be autosomal dominant, autosomal recessive, or X-linked.In addition, most cases of NDM transient subtype (TNDM), which remits within weeks/ months from diabetes outset, are associated with aberrations of chromosome 6q24 (1).NDM genes affect pancreatic beta cell development and/or function that cause severe insulin deficiency, with patients often times presenting with diabetic ketoacidosis (1).Individuals with congenital severe insulin resistance (c.SIR) show syndromic features such as lipodystrophy, acanthosis nigricans, and hirsutism and present with nonketotic diabetes at birth, frequently alternating with hypoglycemia (2).Fasting insulin concentrations >25 μUI/mL (>150 pmolL/L) are considered to be a sign of SIR in lean children/adolescents (1), but in neonates with c.SIR insulin levels in the triple/quadruple-digit range (>100-1000 μU/mL) (2) are often observed at random sampling (neonates are fed every 2-4 hours), along with high C-peptide levels.c.SIR is caused by biallelic, usually recessive, variants in genes that impair insulin action like INSR (2,3).
NDM is not frequent, with an estimated incidence in Europe of about 1:100 000 live births (4), while c.SIR is considered exceedingly rare (an estimated 1:4 000 000 live births for Donohue and Rabson-Mendenhall syndrome) (2).According to the International Society for Pediatric and Adolescent guidelines 2022, all cases with a clinical diagnosis of NDM and c.SIR deserve genetic testing (1).
Until 2013, a relatively short list of genes/chromosomal defects was used to investigate NDM and c.SIR in Europe and the United States, namely, KCNJ11, INS, ABCC8 (both permanent NDM and TNDM), 6q24 (TNDM), and INSR (c.SIR), while some rare, recessive permanent NDM (PNDM) genes (eg, EIF2AK3, GCK, PDX1, PTF1A, GLIS3, others) were screened "on demand," according to the proband's phenotype.By screening the 4 main genes in 27 subjects with NDM identified between 2005 and 2010, we were able to reach a genetic diagnosis in 18 (66%) (4).In the last 15 years, however, the list of genes causing nonautoimmune PNDM has been constantly increasing and now exceeds 25.New genes of autoimmune PNDM have been also discovered, adding to the prototype of this subgroup (ie, FOXP3) (1).In contrast, just 3 genetic defects (6q24, ABCC8, KCNJ11) still account for about 90% of cases with TNDM (1,5).Nevertheless, a few new TNDM genes have been found in recent years (1).
In the last decade, new techniques of DNA sequencing, collectively called "next-generation sequencing" or NGS, progressively replaced the Sanger method in all fields of genetics, including NDM (6,7).By allowing parallel massive DNA sequencing of thousands genes, NGS has proven to be the ideal method to investigate diseases with high genetic heterogeneity, accelerating the identification of pathogenic variants in rare loci.
In this paper we evaluated the impact of NGS on NDM and c.SIR by comparing the 2003-2012 period, when Sanger was the only method in use in Italy, and 2013-2022 period, when NGS progressively became the method of choice.

Materials and Methods
We utilized the definition of NDM and c.SIR described earlier and in (1).NDM was diagnosed in a patient presenting with plasma glucose in the diabetes range within 6 months of age, along with low/undetectable insulin/C-peptide levels.c.SIR was suspected in neonates with diabetes, high insulin (>25 μU/mL), and/or C-peptide levels.

Statistics
Count and rate have been reported for each group.Chi-square analysis was used to compare the subgroups.Statistical significance was set at P < .05.
Informed consent to perform genetic studies for diagnostic purpose was obtained by parents or guardians of the probands.Ethics committee approval was not requested, since the General Authorization to Process Personal Data for Scientific Research Purposes (authorization no.9/2014) declared that retrospective archive studies that use identifier codes, preventing the data from being traced back directly to the data subject, do not need ethics approval.

Results
During
In 2 out of 6 patients with TNDM negative for 6q24 from 2013-2022, KCNJ11 and the rare TNDM genes SLC2A2 and HNF1B, an ABCC8 variant of uncertain significance was identified.In 1 patient the variant of uncertain significance was spontaneous (c.2959T>C, p.Ser987Pro), while in the other (c.157A>T, p.Ser53Cys) it was inherited from the normoglycemic mother.These 2 subjects were included in the TNDM group of unknown etiology (Fig. 2).The remaining 4 infants with TNDM (2013-2022) were negative for ABCC8 and KCNJ11 and lost to follow-up without being tested for 6q24.

NDM and Mode of Inheritance
Most variants in frequent NDM genes (KCNJ11, INS, ABCC8) are dominant and in many patients occur spontaneously (Table 5, second column from the left); however, familial cases with typical vertical transmission are not infrequent (Table 5, third column).Interestingly, in patients with TNDM due to KCNJ11 or ABCC8 mutations, it is not rare to observe a different age at diabetes onset (adolescence, adulthood, ie, not neonatal) in the parent carrying the variant ((6), and this investigation).As already described by others (6), recessive PNDM forms (Table 5) are rarely identified in patients born to nonconsanguineous parents.In our dataset, most individuals with recessive forms of NDM carried homozygous variants, but 2 individuals bore compound heterozygous variants of PDX1 (pancreas hypoplasia) and LRBA (autoimmune case PNDM).7) (9).The single case with the BSCL2 variant somehow resembled INSR-c.SIR because the case presented with hyperglycemia with episodes of hypoglycemia, hirsutism, protruding abdomen, and lipodystrophy, but differed for birth weight (normal) and triglyceride levels (very high in BSCL2, normal-low in INSR; Table 7) and absence of nephrocalcinosis.
Precise genetic diagnosis impacted on therapy of individuals with c.SIR: the patient carrying the BSCL2 variant was started Please note that 6q24 cases have not been reported in the table.Aberrations of 6q24 are usually sporadic, but some specific defects can be inherited.All patients with PNDM or TNDM caused by heterozygous, spontaneous mutations (de novo) have a 50% chance of passing the mutant allele to each child.In this table, 14 cases inherited their variant from a parent; in 2 ABCC8 kindreds, however, the parent carrying the variant was normoglycemic, ie, nonpenetrance was observed.Abbreviations: c.SIR, congenital severe insulin resistance; PNDM, permanent neonatal diabetes mellitus; TNDM, NDM transient subtype.
The Journal of Clinical Endocrinology & Metabolism, 2024, Vol.00, No. 0 on metreleptin (24) with excellent results for glucose and lipid metabolism (manuscript in preparation).In 2 patients with Rabson-Mendenhall syndrome (9) with diabetes of long duration (>3 years) and modest or null response to metformin and insulin therapy, the SGLT2i empagliflozin (5 mg) was introduced as an add-on treatment with good (25) to excellent results (manuscript in preparation).All patients with congenital SIR reported in this investigation bear biallelic, recessive mutations (Table 7).

Discussion
Results of our comparison between 2 decades of investigation on genetic causes of NDM and congenital SIR in Italy shows that NGS made a difference.Pathogenic variants in frequent NDM genes (KCNJ11, INS, ABCC8, 6q24) accounted for 72% and 69.5% in the 2 periods of observation, a similar figure to our previous finding (4).In contrast, during the period 2013-2022, when NGS became increasingly available in genetic laboratories, we identified pathogenic variants in rare recessive or X-linked PNDM genes such as RFX6, PDX1, LRBA, IL2RA, and FOXP3, and a dominant GATA6 variant in a case with pancreas agenesis and heart malformation.In addition, rare causes of TNDM and c.SIR (ie, HNF1B and BSCL2, respectively) were readily detected.These results cannot be attributed to newly identified NDM or c.SIR genes, because 6 of those involved in this study were discovered when Sanger sequencing was the only method used to sequence DNA in Italy, namely, PDX1 (identified in 1997), FOXP3  (34,35).These 2 cases have been considered unsolved and await further investigation to define the etiology of neonatal diabetes.NGS, publicly available database (eg, gnomAD), and functional studies were instrumental to identify and classify pathogenic/likely pathogenic ABCC8 variants in 2 brothers with TNDM that have not been previously resolved.The 2 brothers carried a loss of function variant in 1 allele and the missense variant Thr540Ile in the other.We reasoned that Thr540Ile has a mild activating function and causes TNDM because it is functionally hemizygous.This mechanism of disease has been previously demonstrated in ABCC8-PNDM (36), but to the best of our knowledge, it has never been described in TNDM.
During 2013-2022, we observed an apparent increase in TNDM, likely because screening of blood glucose at birth has become more common with better detection of mild, transient diabetes (5).Nevertheless, many TNDM cases remained unsolved, probably because of a lesser "incentive" to pursue genetic testing in nonsyndromic patients with modest, remitting hyperglycemia treated with short-lived insulin therapy or only rehydration.Of interest, a patient with KCNJ11-TNDM with diabetes onset in 2022 entered remission without any insulin or sulfonylurea treatment, confirming an observation we made in 2 other patients with TNDM associated with KCNJ11 pathogenic variants (5).Therefore, caution should be exerted with the implementation of sulfonylurea therapy before the results of genetic testing because of the risk of hypoglycemia.In addition, this finding reinforces the notion that insulin treatment is an applicable but in some instances dispensable criterion for the diagnosis of TNDM.
NGS was especially useful to identify rare genes of autoimmune PNDM and c.SIR.For instance, patients with FOXP3, IL2RA, and LRBA PNDM may present similar extrapancreatic features (eg, enteropathies, hypothyroidism) (37).In addition, LRBA can cause autoimmune PNDM without syndromic symptoms/signs associated with severe immune dysregulation (38).Simultaneous screening allowed swift identification of autoimmune PNDM genes involved, that in turn lead to a specific therapy (ie, bone marrow transplant).In addition, quick identification of pathogenic variants in RFX6 (34) and PDX1 (previously known as IPF1) (39) along with signs of exocrine insufficiency in PNDM cases with probable pancreatic hypoplasia/agenesis prompted replacement therapy with pancreatic enzymes.Importantly, this intervention was not based on ultrasound imaging of the pancreas.
Our data confirm that INSR mutations are a major cause of c.SIR, with NGS identifying a single patient with the BSCL2 pathogenic variant.In this case, physical examination was puzzling, but 2 clinical features set apart the infant bearing an BSCL2 variant from the INSR group: lack of intrauterine growth restriction and hypertriglyceridemia (Table 7) (40,41).Finally, we found that c.SIR is 12-fold rarer than NDM but about 3 times more frequent than a previous estimate (2).Considering that major causes of NDM in Italy are autosomal dominant genes (KCNJ11, INS, and most of ABCC8 cases) while c.SIR is recessive, this is not an unexpected finding.
A limitation of our study is the small number of patients involved, which does not allow robust conclusions to be drawn about the incidence of rare, recessive forms of PNDM that are common in the Middle East (Fig. 3).For instance, in Abu Dhabi, Turkey, and Quatar biallelic pathogenic variants in the PTF1A enhancer, INS promoter, and EIF2AK3 genes account for 30% to 78% of all cases of PNDM (30,33,42), although we did not identify any such variant.Still, our study illustrates the advantages provided by NGS over Sanger sequencing in terms of time to and precision of genetic diagnosis.We conclude that combining results of massive parallel sequencing with pertinent clinical features of the proband allow prompt confirmation of genetic findings and timely administration of the best available treatments.
CAVIN1, and INSR.Other Italian laboratories involved in NDM/c.SIR genetic screening based in Milan (San Raffaele Hospital), Naples (CEINGE), San Giovanni Rotondo (IRCCS Casa Sollievo della Sofferenza), and Novara (University of Eastern Piedmont) utilized slightly different NGS methods and lists of genes.

Table 2 . Variants identified in common PNDM/TNDM genes KCNJ11, INS, and ABCC8
a Not responsive to sulfonylureas.b Novel variants.