Long-term Multimodal Recording Reveals Epigenetic Adaptation Routes in Dormant Breast Cancer Cells.

Patients with estrogen receptor-positive breast cancer receive adjuvant endocrine therapies (ET) that delay relapse by targeting clinically undetectable micrometastatic deposits. Yet, up to 50% of patients relapse even decades after surgery through unknown mechanisms likely involving dormancy. To investigate genetic and transcriptional changes underlying tumor awakening, we analyzed late relapse patients and longitudinally profiled a rare cohort treated with long-term neoadjuvant ETs until progression. Next, we developed an in vitro evolutionary study to record the adaptive strategies of individual lineages in unperturbed parallel experiments. Our data demonstrate that ETs induce nongenetic cell state transitions into dormancy in a stochastic subset of cells via epigenetic reprogramming. Single lineages with divergent phenotypes awaken unpredictably in the absence of recurrent genetic alterations. Targeting the dormant epigenome shows promising activity against adapting cancer cells. Overall, this study uncovers the contribution of epigenetic adaptation to the evolution of resistance to ETs. SIGNIFICANCE: This study advances the understanding of therapy-induced dormancy with potential clinical implications for breast cancer. Estrogen receptor-positive breast cancer cells adapt to endocrine treatment by entering a dormant state characterized by strong heterochromatinization with no recurrent genetic changes. Targeting the epigenetic rewiring impairs the adaptation of cancer cells to ETs. See related commentary by Llinas-Bertran et al., p. 704. This article is featured in Selected Articles from This Issue, p. 695.

Contribution of pks+ E. coli mutations to colorectal carcinogenesis.

The dominant mutational signature in colorectal cancer genomes is C > T deamination (COSMIC Signature 1) and, in a small subgroup, mismatch repair signature (COSMIC signatures 6 and 44). Mutations in common colorectal cancer driver genes are often not consistent with those signatures. Here we perform whole-genome sequencing of normal colon crypts from cancer patients, matched to a previous multi-omic tumour dataset. We analyse normal crypts that were distant vs adjacent to the cancer. In contrast to healthy individuals, normal crypts of colon cancer patients have a high incidence of pks + (polyketide synthases) E.coli (Escherichia coli) mutational and indel signatures, and this is confirmed by metagenomics. These signatures are compatible with many clonal driver mutations detected in the corresponding cancer samples, including in chromatin modifier genes, supporting their role in early tumourigenesis. These results provide evidence that pks + E.coli is a potential driver of carcinogenesis in the human gut.

Immune selection determines tumor antigenicity and influences response to checkpoint inhibitors.

In cancer, evolutionary forces select for clones that evade the immune system. Here we analyzed >10,000 primary tumors and 356 immune-checkpoint-treated metastases using immune dN/dS, the ratio of nonsynonymous to synonymous mutations in the immunopeptidome, to measure immune selection in cohorts and individuals. We classified tumors as immune edited when antigenic mutations were removed by negative selection and immune escaped when antigenicity was covered up by aberrant immune modulation. Only in immune-edited tumors was immune predation linked to CD8 T cell infiltration. Immune-escaped metastases experienced the best response to immunotherapy, whereas immune-edited patients did not benefit, suggesting a preexisting resistance mechanism. Similarly, in a longitudinal cohort, nivolumab treatment removes neoantigens exclusively in the immunopeptidome of nonimmune-edited patients, the group with the best overall survival response. Our work uses dN/dS to differentiate between immune-edited and immune-escaped tumors, measuring potential antigenicity and ultimately helping predict response to treatment.

The co-evolution of the genome and epigenome in colorectal cancer.

Colorectal malignancies are a leading cause of cancer-related death1 and have undergone extensive genomic study2,3. However, DNA mutations alone do not fully explain malignant transformation4-7. Here we investigate the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,370 samples from 30 primary cancers and 8 concomitant adenomas and generated 1,207 chromatin accessibility profiles, 527 whole genomes and 297 whole transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent somatic chromatin accessibility alterations, including in regulatory regions of cancer driver genes that were otherwise devoid of genetic mutations. Genome-wide alterations in accessibility for transcription factor binding involved CTCF, downregulation of interferon and increased accessibility for SOX and HOX transcription factor families, suggesting the involvement of developmental genes during tumourigenesis. Somatic chromatin accessibility alterations were heritable and distinguished adenomas from cancers. Mutational signature analysis showed that the epigenome in turn influences the accumulation of DNA mutations. This study provides a map of genetic and epigenetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.

Phenotypic plasticity and genetic control in colorectal cancer evolution.

Genetic and epigenetic variation, together with transcriptional plasticity, contribute to intratumour heterogeneity1. The interplay of these biological processes and their respective contributions to tumour evolution remain unknown. Here we show that intratumour genetic ancestry only infrequently affects gene expression traits and subclonal evolution in colorectal cancer (CRC). Using spatially resolved paired whole-genome and transcriptome sequencing, we find that the majority of intratumour variation in gene expression is not strongly heritable but rather 'plastic'. Somatic expression quantitative trait loci analysis identified a number of putative genetic controls of expression by cis-acting coding and non-coding mutations, the majority of which were clonal within a tumour, alongside frequent structural alterations. Consistently, computational inference on the spatial patterning of tumour phylogenies finds that a considerable proportion of CRCs did not show evidence of subclonal selection, with only a subset of putative genetic drivers associated with subclone expansions. Spatial intermixing of clones is common, with some tumours growing exponentially and others only at the periphery. Together, our data suggest that most genetic intratumour variation in CRC has no major phenotypic consequence and that transcriptional plasticity is, instead, widespread within a tumour.

An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia.

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

Circulating tumour DNA sequencing to determine therapeutic response and identify tumour heterogeneity in patients with paediatric solid tumours.

OBJECTIVE: Clinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind. METHODS: To address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of low-frequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood. RESULTS: Analytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89-0.95]) and reproducible (>0.87 [95% CI: 0.87-0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples. CONCLUSIONS: This is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours.

The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition.

Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1-/- mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1-/- cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.

Chemotherapy induces canalization of cell state in childhood B-cell precursor acute lymphoblastic leukemia.

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We, therefore, investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of cell state accounts for a significant component of bottleneck selection during induction chemotherapy.

Mitochondrial Potentiation Ameliorates Age-Related Heterogeneity in Hematopoietic Stem Cell Function.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs in vivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction as Developmental Susceptibility to B Acute Lymphoblastic Leukemia-Associated ETV6-RUNX1.

ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19-IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19-IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state.

Exome sequencing for the differential diagnosis of ciliary chondrodysplasias: Example of a WDR35 mutation case and review of the literature.

Exome sequencing is becoming widely popular and affordable, making it one of the most desirable methods for the identification of rare genetic variants for clinical diagnosis. Here, we report the clinical application of whole exome sequencing for the ultimate diagnosis of a ciliary chondrodysplasia case presented with an initial clinical diagnosis of Asphyxiating Thoracic Dystrophy (ATD, Jeune Syndrome). We have identified a novel homozygous missense mutation in WDR35 (c.206G > A), a gene previously associated with Sensenbrenner Syndrome, Ellis-van Creveld syndrome and Short-rib polydactyly syndrome type V. The genetic findings in this family led to the re-evaluation of the initial diagnosis and a differential diagnosis of Sensenbrenner Syndrome was made after cautious re-examination of the patient. Cell culture studies revealed normal subcellular localization of the mutant WDR35 protein in comparison to wildtype protein, pointing towards impaired protein-protein interaction and/or altered cell signaling pathways as a consequence of the mutated allele. This research study highlights the importance of including pathogenic variant identification in the diagnosis pipeline of ciliary chondrodysplasias, especially for clinically not fully defined phenotypes.

Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome.

EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal dysplasia syndrome, and to gain insight into the pathogenesis of the disorder, we analyzed the localization of EXTL3 in fibroblasts derived from affected individuals and determined glycosaminoglycan concentrations in these cells as well as in urine and blood. We observed abnormal glycosaminoglycan concentrations and increased concentrations of the non-sulfated chondroitin disaccharide D0a0 and the disaccharide D0a4 in serum and urine of all analyzed affected individuals. In summary, we show that biallelic mutations in EXTL3 disturb glycosaminoglycan synthesis and thus lead to a recognizable syndrome characterized by variable expression of skeletal, neurological, and immunological abnormalities.

Phenotypic and Genotypic Characterisation of Inflammatory Bowel Disease Presenting Before the Age of 2 years.

OBJECTIVES: Inflammatory bowel disease [IBD] presenting in early childhood is extremely rare. More recently, progress has been made to identify children with monogenic forms of IBD predominantly presenting very early in life. In this study, we describe the heterogeneous phenotypes and genotypes of patients with IBD presenting before the age of 2 years and establish phenotypic features associated with underlying monogenicity. METHODS: Phenotype data of 62 children with disease onset before the age of 2 years presenting over the past 20 years were reviewed. Children without previously established genetic diagnosis were prospectively recruited for next-generation sequencing. RESULTS: In all, 62 patients [55% male] were identified. The median disease onset was 3 months of age (interquartile range [IQR]: 1 to 11). Conventional IBD classification only applied to 15 patients with Crohn's disease [CD]-like [24%] and three with ulcerative colitis [UC]-like [5%] phenotype; 44 patients [71%] were diagnosed with otherwise unclassifiable IBD. Patients frequently required parenteral nutrition [40%], extensive immunosuppression [31%], haematopoietic stem-cell transplantation [29%], and abdominal surgery [19%]. In 31% of patients, underlying monogenic diseases were established [EPCAM, IL10, IL10RA, IL10RB, FOXP3, LRBA, SKIV2L, TTC37, TTC7A]. Phenotypic features significantly more prevalent in monogenic IBD were: consanguinity, disease onset before the 6th month of life, stunting, extensive intestinal disease and histological evidence of epithelial abnormalities. CONCLUSIONS: IBD in children with disease onset before the age of 2 years is frequently unclassifiable into Crohn's disease and ulcerative colitis, particularly treatment resistant, and can be indistinguishable from monogenic diseases with IBD-like phenotype.

Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis.

BACKGROUND: Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. DESIGN: We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). RESULTS: TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. CONCLUSIONS: Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting.

Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families. METHODS AND RESULTS: Data from whole-exome sequencing in a consanguineous Turkish family, and whole-genome sequencing in the obligate carrier parents of a consanguineous Pakistani family was combined to identify homozygous loss-of-function mutations in ARMC4, segregating in all five affected individuals from both families. Both families carried nonsense mutations within the highly conserved armadillo repeat region of ARMC4: c.2675C>A; pSer892* and c.1972G>T; p.Glu658*. A deficiency of ARMC4 protein was seen in patient's respiratory cilia accompanied by loss of the distal outer dynein arm motors responsible for generating ciliary beating, giving rise to cilia immotility. ARMC4 gene expression is upregulated during ciliogenesis, and we found a predicted interaction with the outer dynein arm protein DNAI2, mutations in which also cause PCD. CONCLUSIONS: We report the first use of whole-genome sequencing to identify gene mutations causing PCD. Loss-of-function mutations in ARMC4 cause PCD with situs inversus and cilia immotility, associated with a loss of the distal outer (but not inner) dynein arms. This addition of ARMC4 to the list of genes associated with ciliary outer dynein arm defects expands our understanding of the complexities of PCD genetics.

Severe resistance to weight gain, lack of stored triglycerides in adipose tissue, hypoglycaemia, and increased energy expenditure: a novel disorder of energy homeostasis.

BACKGROUND: Growth during childhood is a consequence of the equilibrium of energy balance. Obesity results from a shift of the equilibrium towards increased energy intake over expenditure. A clinical description of extreme leanness and failure to thrive secondary to a shift of the equilibrium towards increased energy expenditure over energy intake has not been previously described in the medical literature. SUBJECTS AND METHODS: We report the case of a female child born premature with a birth weight of 1.1 kg who presented with extreme failure to thrive, persistent hypoglycaemia, paucity of fat in the adipose tissue with increased brown fat and increased resting energy expenditure. RESULTS: Complete cessation of weight and height was noted between 3 months to 3.5 years of age. Hypoglycaemia was secondary to depleted energy stores and increased insulin sensitivity. Increased resting energy expenditure was demonstrated on indirect calorimetric assessment. Biopsy of adipose tissue demonstrated paucity of stored fat with increase in brown fat. No gain in weight and height was demonstrated despite high calorie intake of enteral and parenteral feeds. CONCLUSION: We describe a unique case of extreme failure to thrive with increased energy expenditure and severe hypoglycaemia. Unravelling the molecular basis of this novel disorder has the potential to provide insights into the prevention of obesity.

Hyperinsulinaemic hypoglycaemia: genetic mechanisms, diagnosis and management.

Hyperinsulinaemic hypoglycaemia (HH) is due to the unregulated secretion of insulin from pancreatic ß-cells. A rapid diagnosis and appropriate management of these patients is essential to prevent the potentially associated complications like epilepsy, cerebral palsy and neurological impairment. The molecular basis of HH involves defects in key genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A and UCP2) which regulate insulin secretion. The most severe forms of HH are due to loss of function mutations in ABCC8/KCNJ11 which encode the SUR1 and KIR6.2 components respectively of the pancreatic ß-cell K(ATP) channel. At a histological level there are two major forms (diffuse and focal) each with a different genetic aetiology. The diffuse form is inherited in an autosomal recessive (or dominant) manner whereas the focal form is sporadic in inheritance and is localised to a small region of the pancreas. The focal form can now be accurately localised pre-operatively using a specialised positron emission tomography scan with the isotope Fluroine-18L-3, 4-dihydroxyphenyalanine (18F-DOPA-PET). Focal lesionectomy can provide cure from the hypoglycaemia. However the diffuse form is managed medically or by near total pancreatectomy (with high risk of diabetes mellitus). Recent advances in molecular genetics, imaging with 18F-DOPA-PET/CT and novel surgical techniques have changed the clinical approach to patients with HH.

Pancreatic ß-cell KATP channels: Hypoglycaemia and hyperglycaemia.

The pancreatic ß-cell ATP-sensitive K(+) channel (K(ATP) channel) plays a critical role in glucose homeostasis by linking glucose metabolism to electrical excitability and insulin secretion. Changes in the intracellular ratio of ATP/ADP mediate the metabolic regulation of channel activity. The ß-cell K(ATP) channel is a hetero-octameric complex composed of two types of subunits: four inward-rectifying potassium channel pore-forming (Kir6.2) subunits and four high-affinity sulfonylurea receptor 1 (SUR1) subunits. Kir6.2 and SUR1 are encoded by the genes KCNJ11 and ABCC8, respectively. Mutations in these genes can result in congenital hyperinsulinism and permanent neonatal diabetes. This review highlights the important role of the ß-cell K(ATP) channel in glucose physiology and provides an introduction to some of the other review articles in this special edition of the Reviews in Endocrine and Metabolic Disorders.

3-Hydroxyacyl-coenzyme A dehydrogenase deficiency and hyperinsulinemic hypoglycemia: characterization of a novel mutation and severe dietary protein sensitivity.

BACKGROUND: HADH encodes for the enzyme 3-hydroxyacyl-coenzyme A dehydrogenase (HADH) and catalyses the penultimate reaction in the beta-oxidation of fatty acids. All previously reported patients with mutations in HADH gene and hyperinsulinemic hypoglycemia (HH) showed raised plasma hydroxybutyrylcarnitine and urinary 3-hydroxyglutarate. AIMS: The aims of the study were: 1) to report a novel HADH gene mutation not associated with abnormal acylcarnitine or urinary organic acid profile; and 2) to report the novel observation of severe protein-sensitive HH in three patients with HADH gene mutations. RESEARCH DESIGN AND METHODS: The index case presented at 4 months of age with hypoglycemic seizures. Her HH responded to diazoxide, but she continued to have episodes of hypoglycemia even on diazoxide, especially when consuming high-protein foods. RESULTS: Investigations confirmed HH (blood glucose level of 1.8 mmol/liter with simultaneous serum insulin level of 58 mU/liter) with normal acylcarnitines and urine organic acids. Sequencing of the HADH gene identified a homozygous missense mutation (c.562A>G; p.Met188Val). Hydroxyacyl-coenzyme A dehydrogenase activity was significantly decreased compared with controls (index patient, mean +/- sem, 26.8 +/- 4.8 mU/mg protein; controls, 48.0 +/- 8.1 mU/mg protein; P = 0.029) in skin fibroblasts. This patient was severely protein sensitive. Two other children with HH due to HADH gene mutations also demonstrated marked protein sensitivity. CONCLUSIONS: Mutations in the HADH gene are associated with protein-induced HH, and patients with HH due to HADH gene mutations may have normal acylcarnitines and urine organic acids.