Lethal neonatal acidosis: Multiomic investigation of a novel HIBCH variant as the underlying cause

Abstract

HIBCH (3-Hydroxyisobutyryl-CoA hydrolase) deficiency is a rare, autosomal recessive inborn error of metabolism caused by pathogenic variants in HIBCH and typically presenting in the first year of life with hypotonia, seizures, global developmental delay, poor feeding, and ataxia. Biochemical abnormalities such as lactic acidosis and hyperammonemia may also be seen due to disruption of mitochondrial function, and the diagnosis may also be suspected by the presence of elevated hydroxy-C4-carnitine (C4-OH) detected from a blood sample with a definitive diagnosis obtainable by genetic analysis. We describe a neonate with mild hypotonia at birth who rapidly developed a severe metabolic acidosis, with her venous pH reaching a nadir of 6.374 within hours of life and death occurring within 15 h of life despite supportive measures. A genomic autopsy was undertaken using a blood sample saved prior to the neonatal death. Postmortem trio exome sequencing of the neonate and both parents revealed the neonate to be homozygous for a novel variant in HIBCH predicted to impact splicing, presumably resulting in severe deficiency of HIBCH enzyme activity. As both parents were carriers of the causal variant, anticipatory guidance was provided for risk reduction in future pregnancies. This case highlights the importance of comprehensive postmortem evaluation to evaluate severe, neonatal lethal conditions.

1. Introduction

HIBCH (3-Hydroxyisobutyryl-CoA hydrolase) deficiency (OMIM #250620), first reported in 1982, is an ultrarare, autosomal recessive disease [1], with a prevalence of 1 in 551,545 Europeans and 1 in 127, 939 South Asians [2]. 3-Hydroxyisobutyryl-CoA hydrolase is a nuclear encoded mitochondrial enzyme that is responsible for the hydrolysis of 3-hydroxyisobutyryl-CoA to 3-hydroxy-isobutyric acid as the fifth step in valine catabolism [3,4]. Decreased HIBCH enzyme activity thus produces an accumulation of toxic valine metabolites made upstream in the metabolic pathway, leading to oxidative stress and impaired mitochondrial enzyme activity, ultimately causing the damaging multi-organ effects seen in this disease [1,5].

The typical presentation of HIBCH deficiency is within the first year of life with motor and neurodevelopmental delay, hypotonia, ataxia, dystonia, seizures, poor feeding, optic nerve atrophy and organic aciduria. Magnetic resonance imaging (MRI) of the brain typically reveals abnormalities in deep gray matter, the bilateral globus pallidus and cerebral peduncles, as well as white matter atrophy [[5], [6], [7]]. On brain magnetic resonance spectroscopy, an elevation of plasma 3-hydroxyisobutyryl carnitine is often seen, while elevations in lactate and pyruvate levels are less common [[5], [6], [7]]. Due to impaired mitochondrial function caused by impaired HIBCH function, the clinical presentation and MRI findings of HIBCH deficiency closely resemble Leigh syndrome: a neurometabolic syndrome caused by defects in oxidative phosphorylation [5,7,8]. A diagnosis of HIBCH deficiency may be suspected in the setting of elevation of plasma 3-hydroxyisobutyryl carnitine, detected as hydroxy-C4-carnitine (C4-OH) in a blood sample or dried blood spot from a newborn state screen [6], and of 2-methyl-2,3 dihydroxybutyric acid and methacrylyl-CoA metabolites found via urine organic acid analysis [1]. Measurement of HIBCH enzymatic activity or a molecular genetic analysis of the causal gene, HIBCH, can be used to confirm the diagnosis, with the latter technique representing a less-invasive approach as a tissue sample is required for enzyme analysis whereas DNA from blood or a buccal swab may be used for molecular diagnosis.

In this report we describe an infant who died at 15 h of life after presenting with hypotonia and profound metabolic acidosis within hours of birth. Diagnosis of HIBCH deficiency was made via postmortem trio exome sequencing with supporting evidence identified via RNA sequencing in addition to tandem mass spectrometry on newborn screen.

2. Case presentation

2.1. Clinical presentation

A female neonate was born at 39 weeks and 6 days of gestation to a 28-year-old gravida 3, para 1011 mother. The neonate was born via spontaneous vaginal delivery after induction of labor for gestational hypertension. The pregnancy and delivery were otherwise without significant complications, although the mother presented late to prenatal care in the setting of seeking refugee status outside of her home country of Haiti. The infant was mildly hypotonic at birth with Apgar scores of 8 at one minute (−1 for color, −1 for tone) and 9 and 5 min (−1 for color). By one hour of life, she was admitted to the level III NICU at her birth hospital in the setting of significant hypotonia and poor reflexes. The hypotonia was at first attributed to hypoglycemia as her initial blood glucose level was 16 mg/dL (reference range 41-199 mg/dL), treated per institutional protocols with a glucose gel (40 % glucose) and syringe feed of formula. She did require multiple glucose gel treatments and was ultimately started on continuous intravenous infusion with dextrose. Laboratory findings at 6 h of life were notable for an anion gap metabolic acidosis (pH 7.1, bicarbonate level < 10 mEq/L, anion gap of 23). At 10 h of life, she became acutely hypoxic, requiring endotracheal intubation. Repeat venous blood gas sampling at that time revealed a pH of 6.77 and bicarbonate of 6 mEq/L with lactic acid of 17 mmol/L and serum ammonia of 96 μmol/L. Due to her severe metabolic acidosis and elevated lactic acid, there was high suspicion for a congenital mitochondrial disorder.

The neonate was thus transferred emergently from the birth hospital to a level IV NICU. She developed worsening hypotension en route for which she received epinephrine, although her condition continued to deteriorate and she ultimately developed cardiac arrest and required chest compressions for 7 min before successful return of circulation was obtained. Repeat labs upon admission to the level IV NICU demonstrated worsening metabolic acidosis (pH nadir of 6.374), worsening lactic acidosis (to 22 mmol/L), hyperammonemia (378 μmol/L increasing to 536 μmol/L), and severe coagulopathy. Fluid boluses, bicarbonate boluses, and multiple blood products were administered without improvement, and two hours after admission, the infant again went into cardiac arrest. Attempts at resuscitation, including multiple doses of epinephrine, calcium gluconate and sodium bicarbonate boluses, were unsuccessful and the neonate died at 15 h of life.

Post-mortem dysmorphology examination at the time of autopsy was notable for a low anterior hair line, depressed nasal bridge, upturned nares, and a long and smooth philtrum with a thin upper lip. There was no evidence of any internal structural anomalies. There was evidence of physiologic stress, including thymic involution, normoblastemia, and stress-related changes in the adrenal glands. There was also presence of Candida albicans in the blood cultures taken at the time of the autopsy, however cultures obtained premortem were negative and thus this was thought to be a postmortem contaminant. A frozen tissue sample from liver was saved for potential future investigations.

2.2. Genetic testing and results

The family was enrolled into a research protocol with the Manton Center for Orphan Disease Research. Through this protocol, trio exome sequencing was performed via the Children's Hospital Rare Disease Cohorts initiative [9] and revealed a homozygous splice-impacting variant in HIBCH: (NM_014362.3) c.305–2 A > T (g.190,290,487). This variant is predicted by spliceAI [10] and Pangolin [11] to result in loss of the canonical exon 5 splice acceptor, and a gain of a cryptic splice acceptor 14 bp from the variant, causing an in-frame deletion of 12 bp. The variant has a frequency of 9 heterozygotes in gnomAD (all in people of African ancestry) with no homozygotes noted. This finding was validated in a CLIA-certified laboratory, where both parents were confirmed to be carriers of the variant, and it was formally classified as a variant of uncertain significance.

2.3. RNA sequencing and results

To further investigate the impact of the splicing variant, RNA sequencing from frozen liver tissue obtained postmortem was performed at the Broad Institute of MIT and Harvard. The transcriptome product combines poly(A)-selection of mRNA transcripts with a strand-specific cDNA library preparation, with a mean insert size of 550 bp. Libraries were sequenced on the HiSeq 2500 platform with 150 bp paired-end reads, to a depth of 149 million reads. The sequencing data were processed with a pipeline adapted from one developed by the GTEx Consortium [12]. Briefly, FASTQ files were aligned to the GRCh38 reference sequence using STAR-2.6.1b in 2-pass mode, and duplicates were marked with Picard. RNA-seq data were de-multiplexed and each sample sequence data aggregated into a single Picard BAM file. Post-sequencing quality control was performed using RNASeQC2 [13]. Analysis and visualization was performed using IGV.js tracks [14] within TGG-viewer (https://github.com/broadinstitute/tgg-viewer). Review of the splice junction predicted to be impacted by this variant confirms the prediction of a cryptic splice acceptor in exon 5, resulting in an inframe deletion of 12 base pairs (Fig. 1 A). Additionally, RNA-seq shows a cryptic out-of-frame exon (89 bp) in intron 6 (g.190,272,934-190,273,022) (Fig. 1B), predicted to result in premature truncation (Fig. 1C). The total predicted protein length is 148 amino acids (including the 4 amino acid truncation in exon 5, and addition of 6 amino acids from cryptic exon 7 prior to the stop codon), compared to a normal length of 386 amino acids (MANE Select transcript). In gnomAD SV v4.1.0 there are 3 individuals with African ancestry who have a 3.9 kb deletion in intron 6 (chr2:190,276,671-190,280,595). We speculate that this deletion might be in cis with the c.305–2 A > T variant, as both are seen only in individuals with African ancestry, and a combination of both alleles on the same haplotype may account for the cryptic splicing seen here. Long read genome sequencing would be required to definitively phase this deletion with the c.305–2 A > T variant.

Fig. 1.

RNA sequencing results from the proband compared to GTEx control data.

A Exon 5 of HIBCH. Blue bar indicates the reference exon 5, and the pink bar shows the RNA-seq junction read that spans exon 5, demonstrating a 12 bp truncation and loss of the canonical splice acceptor.

B Sashimi plot of the splicing pattern seen in RNA-seq in the proband. A cryptic 89 bp exon is seen in intron 6. Red bar indicates a 3.9 kb deletion seen in 3 individuals in gnomAD SV v4.1.0 with African ancestry.

C Cryptic exon within canonical intron 6 of HIBCH. Predicted to result in premature truncation.

2.4. Newborn screen follow-up

Although a formal report was not issued for this neonate due to her demise within 24 h of age, tandem mass spectrometry data from the dried blood spot sample taken upon admission to the level IV NICU was obtained and analyzed. Notable findings include C4-OH/C3 ratio 0.91 [+7.6 SD]; C4 1.09 μmol [+ 3.6 SD]; C5—0H 0.45 μmol [+ 5.1 SD]; C4-OH 0.52 μmol [+ 3.2 SD], which would have prompted diagnostic testing for an inborn error of metabolism in the branched chain amino acid pathways. This pattern is thus consistent with the diagnosis of 3-Hydroxyisobutyryl-CoA hydrolase deficiency.

Although a formal report was not issued for this neonate due to her demise within 24 h of age, tandem mass spectrometry data from the dried blood spot sample taken upon admission to the level IV NICU was obtained and analyzed. The concentration of the relevant markers and the values of the ratios utilized by the screening program in screening for C4-OH related disorders (with their respective z-scores and cut-offs for the normal population shown in parenthesis) were: C4-OH/C3 ratio 0.91 [+7.6 SD; mean 0.07; cut-off <0.5]; C4-OH 0.52 μmol [+ 3.2 SD; mean 0.11; cut-off <0.5]; C4 1.09 μmol [+ 3.6 SD; mean 0.31; cut-off <1.8]; C5-OH 0.45 μmol [+ 5.1 SD; mean 0.11; cut-Off <0.8]. This metabolic profile would have prompted diagnostic testing for an inborn error of metabolism in the branched chain amino acid pathways, and the pattern is supportive of a diagnosis of 3-Hydroxyisobutyryl-CoA hydrolase deficiency.

2.5. Return of results and follow-up

The parents were informed of their carrier status for this novel variant and were counseled that this variant, or the haplotype including this variant, was thought clinically to be responsible for the severe acidosis and multi-organ failure seen in their daughter. They subsequently had a spontaneous pregnancy that was tested via chorionic villous sampling, and the fetus was found to be a heterozygous carrier of the HIBCH variant.

3. Discussion

We present a severe, neonatal-onset case of HIBCH deficiency, diagnosed postmortem by exome sequencing following the death of the neonate at 15 h of life. HIBCH deficiency may present with a wide range of symptoms and clinical outcomes, with the prior earliest reported death in the literature at 36 h of life, also in the setting of severe anion gap metabolic acidosis, although some affected individuals have lived into adulthood with mild symptoms [15,16]. Neuroimaging and enzyme analysis (which can be performed via cultured fibroblasts from skin biopsy) were not able to be completed in our case due to the clinical instability and death of the infant prior to imaging or sample collection, but a saved blood sample prior to death allowed for genomic autopsy via trio exome sequencing, the dried blood spot obtained for the newborn screen allowed for metabolomic investigation, and postmortem liver biopsy facilitated transcriptomic evaluation.

The infant described presented shortly after birth with hypotonia after an uncomplicated pregnancy and vaginal birth. This was the first symptom noted in the infant and it was originally attributed to her concurrent, severe hypoglycemia. Based on previously reported cases, hypotonia is a common symptom seen in the majority of infants with HIBCH deficiency, however due to the rapid progression of this infant's case the other common symptoms of poor feeding, developmental delay, and regression were not observed and her degree of illness precluded evaluation by electroencephogram (EEG) for seizure activity. In addition, congenital malformations have been seen in other reported cases of HIBCH deficiency, similarly to other mitochondrial disorders [1,7,15,[17], [18], [19]]. While the infant was noted to have minor dysmorphic facial features, on postmortem exam, no internal abnormalities were noted. Although the elevated C4-OH seen on the newborn dried blood spot sample sent to the state lab may have suggested an inborn error of metabolism, this finding is not specific for HIBCH deficiency, and the newborn died before this result would have been reported.

This case therefore supports the diagnostic and clinical value of genomic autopsy, or postmortem genome-wide sequencing such as exome or genome sequencing to identify rare Mendelian conditions leading to death. Although the severity of the metabolic acidosis strongly suggested a genetic metabolic condition, there were no clinical findings pointing to this specific diagnosis. While a postnatal diagnosis of HIBCH deficiency would be unlikely to alter the course for this neonate, where the variants identified are presumed to severely depress enzyme activity, the finding of this autosomal recessive disorder is impactful for the parents. Prenatal diagnosis (as was pursued in this case) or pre-implantation diagnosis following in vitro fertilization may be offered to avoid having another affected child. Although there are no precision therapies available for this disorder either pre- or postnatally, aside from the use of a valine-restricted diet in affected children [20], this is a future possibility for which molecular diagnosis will be critical to determine eligibility. Importantly, the rapid decline of the neonate impeded access to clinical genomic sequencing, and it was only through a research genomic autopsy study that sequencing could be performed, enhanced by transcriptomic and metabolic evaluation. This also highlights the value of genomic autopsy in revealing the molecular underpinnings of lethal phenotypes that otherwise would be missed in standard clinical practice.

CRediT authorship contribution statement

Sonali Patel: Writing – review & editing, Writing – original draft, Investigation. Muhammad Zain-ul-abideen: Writing – review & editing, Writing – original draft, Investigation. Genevieve Guyol: Writing – review & editing, Data curation, Conceptualization. Lance H. Rodan: Writing – review & editing, Investigation. Casie A. Genetti: Writing – review & editing, Project administration, Investigation, Formal analysis. Amy Z. Ren: Writing – review & editing, Investigation. Philip Connors: Writing – review & editing, Investigation. Patricia Davenport: Writing – review & editing, Investigation. Ruby Bartolome: Writing – review & editing, Investigation, Conceptualization. Inderneel Sahai: Writing – review & editing, Formal analysis. Vijay S. Ganesh: Writing – review & editing, Formal analysis. Monica H. Wojcik: Writing – review & editing, Writing – original draft, Formal analysis, Conceptualization.

Informed consent

Written informed consent was obtained from the patients for their anonymized information to be published in this article. All procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000.

Funding information

MHW is supported by NICHD K23 HD102589. VSG is supported by NIAMS K23AR083505, and by the BroadIgnite Award. RNA sequencing and analysis was provided by the Broad Institute Center for Mendelian Genomics (Broad CMG) and was funded by UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute) and U01HG011755 (GREGoR consortium). The content is solely the resposibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Declaration of competing interest

No authors have conflicts of interest to declare.

Data availability

Genomic data are available for sharing upon reasonable request and pending execution of a data transfer agreement.