sequencing was undertaken after SNP analysis revealed a large shared region of homozygosity spanning the HADH
locus in six unrelated consanguineous probands. We identified mutations in three of these patients and in a further two probands from known consanguineous families (28% positive). These results prompted us to sequence HADH
in the remainder of our cohort, and homozygous mutations were identified in a further five probands who were not reported as being consanguineous. However, these patients were all referred from countries with high rates of consanguineous marriages. Only one patient with compound heterozygous mutations was identified. In total, HADH
mutations were identified in 11/115 (10%) patients with diazoxide-responsive HH of unknown etiology. Our study increases the number of patients reported with HADH
HH from five probands (4
) to 16.
In one patient with a maternally inherited frame shift mutation, extensive studies to search for a second mutation of paternal origin were undertaken and a novel variant was found in intron 6 (corresponding to RefSeq NM_005327.2). In silico
splicing prediction programs suggest that this variant will not alter splicing. However, at least eight transcript variants are predicted to exist for HADH
, and this substitution results in a missense mutation in exon 6 of a variant transcript (cDNA accession number BI826991
) (), which is predicted to encode a protein with an NAD-binding and a C-terminal domain. Although further studies are required to assess the significance of this transcript, its detection in tissues including pancreas and islets suggests that it may be important. While the pathogenicity of the variant is currently unproven, the identification of a frame shift mutation on the opposite allele is consistent with a diagnosis of recessively inherited HH resulting from a HADH
In keeping with previous reports a range in birth weights and ages at diagnosis of HH were observed in the patients with HADH
). Interestingly, none of the patients were reported to have abnormalities in plasma acylcarnitines or urine organic acids, a phenotype reported in 3/5 published probands. While the absence of abnormal acylcarnitines and urine organic acids in these patients may be attributable to limitations in laboratory analysis, it is possible that the phenotype is mutation-dependent because none of the mutations identified in this cohort have been found in individuals with abnormal acylcarnitines and urine organic acids. It is unlikely, however, that the disease spectrum reflects the severity of the mutation as the majority of patients with isolated HH have null mutations.
A number of studies have demonstrated that HADH has a pivotal role in regulating insulin secretion (13
). Most recently a study by Li et al.
) examined the mechanism of insulin dysregulation in mice with a knock-out of the hadh
gene. Pull-down experiments demonstrated protein–protein interactions between HADH and glutamate dehydrogenase (GDH), and studies on isolated islets showed in increased in the affinity of GDH for its substrate α-ketoglutarate. It is therefore likely that HADH
mutations cause HH by activation of GDH via loss of inhibitory regulation of GDH by HADH. This finding is of particular interest as activating mutations in GLUD1
, which encodes GDH, are a common cause of hyperinsulinism and hyperammonemia (2
In conclusion we have shown that mutations in HADH
account for 10% of cases with diazoxide-responsive HH without a mutation in the known genes. This study takes the number of HADH
mutations identified in our cohort to 12 (7
), with a prevalence similar to that for HH attributable to HNF4A
) mutations. We recommend that analysis of the HADH
gene is considered in all patients with diazoxide-responsive HH who originate from known consanguineous pedigrees, isolated populations, or countries where inbreeding is frequent, regardless of whether there is evidence of abnormal fatty acid oxidation.