We have shown that increased birthweight and macrosomia are common features of patients with HNF4A
mutations and, in addition, that some individuals with HNF4A
have neonatal hypoglycaemia. Although in young adults the same genetic defect results in diabetes due to reduced insulin secretion [15
], we have shown that the mechanism for the phenotype in newborns is likely to be increased insulin secretion in utero and in the neonatal period. This is supported by hyperinsulinaemia in some affected infants with HNF4A
mutations, and studies in mice with β-cell deletion of Hnf4a
clearly show hyperinsulinaemia in utero and hyperinsulinaemic hypoglycaemia in the early neonatal period.
HNF4A-mutation carriers are, on average, 790 g heavier than their family members who do not carry the mutation, and 56% are born with macrosomia (>4,000 g). The increase in birthweight is similar to that seen in the offspring of patients with maternal diabetes which is the commonest recognized cause of macrosomia. Forty-six percent of children with an HNF4A mutation born to affected fathers had macrosomia. This is a clear example that macrosomia may result from foetal genetics as well as from the maternal intra-uterine environment. Consideration of this should be taken into account when determining macrosomia risk, and we recommend that, in addition to maternal diabetes, a history of young-onset non-insulin-requiring paternal diabetes should prompt assessment of foetal size.
We have described eight patients with HNF4A mutations who had one or more episodes of hypoglycaemia in the neonatal period; there was hyperinsulinaemia in all three patients who were tested. Five patients required treatment with intravenous glucose only, with resolution within 1 mo; this finding was consistent with a transient hyperinsulinaemia. Two patients had more persistent hypoglycaemia which responded well to treatment with diazoxide and chlorothiazide and subsequently resolved. Therefore, the loss of HNF4A function causes a relatively mild form of hyperinsulinaemic hypoglycaemia that is transient and diazoxide-responsive. Transient hypoglycaemia is often not investigated and as a result is understudied. We propose that neonates presenting with hypoglycaemia who have a father with diabetes, or a mother with young-onset non-insulin-requiring diabetes, should be screened for HNF4A mutations. However, three out of the five unselected HNF4A-mutation carriers with neonatal hypoglycaemia presented before their respective parent developed diabetes. Therefore, we also suggest that HNF4A mutations should be considered in any child with persistent hypoglycaemia (>24 h).
We encountered two problems resulting from the retrospective nature of this study. Firstly, hospital records were not readily available so birthweight and gestational age were ascertained by parental recall in the majority of cases. However, all this data collection was done blind to genotype and therefore any error should apply to both offspring groups. Secondly, the hypoglycaemia was often not well investigated at the time of presentation, presumably because of its transient nature. Hence, hyperinsulinaemia at birth was looked for only in three out of the eight patients presenting with hypoglycaemia, and other causes of hyperinsulinaemia were not excluded. It also means that we are uncertain whether the 46 patients with HNF4A mutations in whom hypoglycaemia was not described had undetected hypoglycaemia or were not hypoglycaemic. A prospective study of neonates born to HNF4A-mutation carriers is required for a complete assessment of the hyperinsulinaemic hypoglycaemia seen in these patients.
The increased birthweight and risk of macrosomia in HNF4A-mutation carriers is likely to be secondary to foetal hyperinsulinaemia. Although no measures of foetal insulin or cord insulin were available to confirm this mechanism in humans, two lines of evidence support it. Firstly, in humans, we have documented hyperinsulinaemia soon after birth in the three patients in whom it was tested, and hypoglycaemia in eight. Secondly, we have shown that mice lacking pancreatic Hnf4a have increased insulin concentrations in utero, and hyperinsulinaemic hypoglycaemia as newborns.
The increased birthweight and neonatal hypoglycaemia in HNF4A-
mutation carriers seems paradoxical for a gene that is associated with a β-cell–deficient form of young-onset diabetes [15
], particularly as the decreased β-cell function has been explained by decreased expression of pancreatic β-cell genes involved in glucose metabolism [29
]. It is in contrast to other monogenic causes of diabetes—for example, GCK
], or activating KCNJ11
] and ABCC8
] where birthweight is reduced. In these cases, the low insulin secretion that causes diabetes later is associated with decreased insulin-mediated foetal growth. In HNF4A
-mutation carriers, in contrast, there would need to be a switch from increased insulin secretion in utero and neonatal life to decreased insulin secretion in later life. The closest example of this is patients with hyperinsulinaemia of infancy due to recessive and dominant mutations in KATP
channel subunits, who have a high rate of diabetes at long-term follow-up even when they do not receive pancreatic surgery [8
]. It has been postulated that diabetes in SUR1-deficient patients reflects increased apoptosis, in addition to abnormal regulation of secretion due to lack of KATP
]. Compared to SUR1 deficiency, HNF-4α deficiency results in less severe hyperinsulinism, yet gives rise to a more highly penetrant and severe diabetic phenotype. It is interesting, however, that of the eight patients who developed established hypoglycaemia at birth, five developed diabetes by the age of 14 y (mean age 12.4 y), suggesting a possible earlier progression to diabetes in this group.
Two recent studies surprisingly showed that β-cell Hnf4a
deficiency does not cause diabetes in mice [21
]. One study paradoxically reported mildly reduced blood glucose and increased insulin levels in adult β-cell Hnf4a
-deficient mice, and ascribed this to diminished expression of Kcnj11
encoding the KATP
channel subunit Kir6.2 [21
]. Another study failed to confirm abnormal blood glucose and insulin levels, and reported normal Kcnj11
]. This discrepancy, together with the unexpected failure to develop hypoinsulinaemic diabetes, led us to question whether hyperinsulinaemia was an important feature of Hnf4a
deficiency. In the current study, we have shown that, in parallel with the human findings, Hnf4a
–deficient mice exhibit hyperinsulinaemia in the foetal and neonatal stage, as well as overt neonatal hypoglycaemia as opposed to only mildly reduced glucose at later ages as recently reported [21
]. Importantly, our studies showed no abnormal expression of Kcnj11
. While discrepancies in phenotype might be explained by small differences in genetic background, the current data suggest that the hyperinsulinaemic phenotype in Hnf4a
deficiency is not related to KATP
Further studies will need to address how Hnf-4α-dependent transcription in β-cells is linked to the dual phenotype reported here. Large-scale profiling shows that Hnf-4α-deficient β-cells exhibit abnormal expression of more than 10% of all islet genes (unpublished observations), many of which need to be examined as plausible candidates for the HNF4A
-deficient hypersecretory phenotype. It is tempting to hypothesize that the initial defect that causes β-cell hypersecretion might eventually lead to β-cell exhaustion and diabetes, in analogy to what is observed in some patients with SUR1 mutations as described above [8
]. However, the broad transcriptional phenotype of Hnf4a-
deficient mice offers an alternative potential explanation, whereby one HNF-4α-dependent gene-expression defect causes hypersecretion early in life, while a separate gene-expression defect is responsible for the development of severe β-cell failure several years after birth.
The birthweight and incidence of hypoglycaemia in heterozygous HNF1A
-mutation carriers were not different from their unaffected family members. This finding suggests that foetal insulin secretion is not increased in HNF1A
-mutation carriers. Previous data had supported a common phenotype of HNF1A-
-mutation carriers, due to a regulatory transcription factor circuit in the β-cell with positive feedback on expression between HNF-1α and HNF-4α [18
]. Our findings suggest that, at least in foetal life, there are clearly independent functions of these two transcription factors in the foetal β-cell. Our data therefore suggest that, in humans, the proposed transcription-factor network is not critically required in foetal development and early post-natal life.
To conclude, we have shown that heterozygous HNF4A
mutations are associated with a 790 g increase in birthweight, on average, and considerable risk of macrosomia. The increased birthweight is probably mediated by increased foetal insulin secretion and, in some cases, is associated with transient neonatal hyperinsulinaemia. Because HNF4A
deficiency is also known to cause hypoinsulinaemic diabetes, this study shows for the first time that HNF-4α has dual opposing roles in the β-cell during different periods of life. This study also has important implications for clinical practice (see Box 1
). Firstly, pregnancies where a parent is known to have an HNF4A
mutation should be monitored closely during pregnancy and the immediate post-natal period to minimize complications of macrosomia and neonatal hypoglycaemia. Secondly, neonates with transient or persistent hypoglycaemia and/or macrosomia and a family history of young-onset diabetes should be considered for HNF4A
molecular genetic testing. Thirdly, since the foetal genotype has a considerable impact on determining birthweight, in addition to maternal factors, paternal factors (including history of diabetes) should be considered when assessing macrosomia risk.
Box 1. Practical Clinical Points for Diagnosis and Management of Patients with HNF4A Mutations
Management of pregnancy in families known to have diabetes due to HNF4A mutations
Serial antenatal scans should be performed in any pregnancy in which the father or mother is a mutation carrier and early induction of labour is considered. This is true when the mother does not have diabetes, but particularly applies when the mother has diabetes or impaired glucose tolerance in pregnancy.
All offspring of pregnancies where the father or mother is an HNF4A-mutation carrier should be tested for neonatal hypoglycaemia at birth and also 24 h after birth.
In families where there is autosomal dominant inheritance of young-onset diabetes with features consistent with a diagnosis of MODY, details of birthweight and neonatal hypoglycaemia should be specifically asked for.
When macrosomia or neonatal hypoglycaemia (>24 h) is described, HNF4A should be sequenced before other genes when performing diagnostic genetic testing.
Diagnosing and managing neonatal hypoglycaemia
HNF4A should be sequenced in children with neonatal hypoglycaemia, particularly if the hypoglycaemia is relatively mild or transient, or if a family member (parent or grandparent) has young-onset diabetes (<35 y).
In patients diagnosed as having HNF4A, resolution of symptoms should be expected in the first year, but diabetes should be expected to develop in adolescence or in early adulthood and should be screened for annually after the age of 10 y.
HNF4A should be sequenced as part of an investigation of unexplained macrosomia, particularly when the macrosomia is extreme, is accompanied by hypoglycaemia, or there is a family history of early-onset diabetes.