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HADHA and HADHB gene associated phenotypes - Identification of rare variants in a patient cohort by Next Generation Sequencing
Molecular and Cellular Probes 44 (2019) 14–20
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HADHA and HADHB gene associated phenotypes - Identification of rare variants in a patient cohort by Next Generation Sequencing
T
Isabel Diebolda, Ulrike Schöna, Rita Horvathc, Oliver Schwartzd, Elke Holinski-Federa, Heike Kölbele, Angela Abichta,b,∗ a
Medical Genetics Center, Munich, Germany Department of Neurology, Friedrich-Baur-Institute, Klinikum der Ludwig-Maximilians-University, Munich, Germany c Department of Clinical Neuroscience, University of Cambridge, Cambridge, UK d Department of Neuropediatrics, University Children's Hospital Muenster, Muenster, Germany e Department of Pediatric Neurology, Developmental Neurology and Social Pediatrics, University of Essen, Germany b
ARTICLE INFO
ABSTRACT
Keywords: Mitochondrial trifunctional protein HADHA HADHB Next generation sequencing Neuropathy Metabolic myopathy
The heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits harbours three enzymes that each perform a different function in mitochondrial fatty acid β-oxidation. Pathogenic variants in the MTP genes (HADHA and HADHB) cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by phenotypic heterogeneity ranging from severe, early-onset, cardiac disease to milder, later-onset, myopathy and neuropathy. Since metabolic myopathies and neuropathies are a group of rare genetic disorders and their associated muscle symptoms may be subtle, the diagnosis is often delayed. Here we evaluated data of 161 patients with myopathy and 242 patients with neuropathy via next generation sequencing (NGS) and report the diagnostic yield in three patients of this cohort by the detection of disease-causing variants in the HADHA or HADHB gene. The mitigated phenotypes of this treatable disease were missed by the newborn screening, highlighting the importance of phenotype-based NGS analysis in patients with rare and clinically very variable disorders such as MTP deficiency.
1. Introduction Mitochondrial trifunctional protein (MTP) is a large heteromultimeric enzyme that catalyzes the three final steps of long-chain mitochondrial fatty acid β-oxidation, providing an important energy source for skeletal muscle and the heart. MTP is composed of four hydroacyl-CoA dehydrogenase-α (HADHA) and four hydroacyl-CoA dehydrogenase-β (HADHB) subunits encoded by the HADHA and HADHB genes. The genes HADHA and HADHB are located in a head-to-head configuration on chromosome 2p23.3 and share a common promoter. The HADHA gene contains the information for the long-chain enoylCoA hydratase (LCEH) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), whereas the HADHB encodes long-chain ketoacylCoA thiolase (LCKT) activity. To date, two different biochemical phenotypes of defects in the MTP complex are known, the isolated LCHAD deficiency (LCHADD) (OMIM# 609016) and generalized MTP deficiency (OMIM# 609015). Both, isolated LCHAD and MTP deficiency, lead to an accumulation of toxic β-oxidation intermediates causing acute symptoms as well as long-term complications with particularly high morbidity and mortality. Clinical symptoms mainly
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develop during periods of illness or fasting and affect preferably the heart and the skeletal muscle. Thus, clinical manifestations of LCHAD/MTP deficiency can vary from early-onset cardiomyopathy, hypoglycemia, hepatopathy, coma, and sudden infant death syndrome (SIDS) to later onset myopathy, neuropathy and pigmentary retinopathy [1,2]. More than 60% of surviving individuals with LCHAD/MTP deficiency present with myopathy, about 20% develop a slowly progressing peripheral neuropathy, and more than 40% have pigmentary retinopathy [1]. Adults, adolescents or older children may develop recurrent rhabdomyolysis during illness. If LCHAD/MTP deficiency is diagnosed in time, outcome can be favorable, prompting the implementation in newborn screening programs [3]. Acylcarnitines are determined by tandem mass spectrometry with 3-hydroxypalmitoylcarnitine (C16eOH) and 3-hydroxyoleoylcarnitine (C18:1OH) as primary biomarkers [4]. However, there are limitations of newborn screening for LCHAD/MTP deficiency and clinical symptoms of children unidentified as newborns may be subtle. Deficiencies of MTP/LCHAD are autosomal recessively inherited disorders with an estimated frequency of 1:140,000. A prevalent missense mutation (c.1528G > C, (p.Glu510Gln)) in the LCHAD domain of the HADHA gene can be detected in approximately 90% of LCHAD-
Corresponding author. Medical Genetics Center, Munich, Germany. E-mail address: [email protected] (A. Abicht).
https://doi.org/10.1016/j.mcp.2019.01.003 Received 13 December 2018; Received in revised form 17 January 2019; Accepted 20 January 2019 Available online 22 January 2019 0890-8508/ © 2019 Elsevier Ltd. All rights reserved.
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deficient alleles, and patients homozygous for this variant show widely different phenotypes, thus making molecular screening for LCHADD important and feasible. Compared to LCHADD, the molecular basis of MTP deficiency is heterogeneous and different variants have been identified in both HADHA and HADHB genes with poor genotype–phenotype correlation. To date the Human Gene Mutation Database (HGMD) reports 64 HADHA and 60 HADHB gene mutations, many of them have not been functionally characterized. Treatment of LCHAD/MTP deficiency involves avoidance of prolonged fasting, dietary fat restriction, medium chain triglycerides (MCT) supplementation. A low-fat diet and medium chain triglycerides (MCT) supplementation decrease plasma hydroxyl acylcarnitine levels, and most patients remain healthy without metabolic decompensation but still require supplementation with essential fatty acids and fat-soluble vitamins [5,6]. Further treatment options such as anaplerotic therapy with heptanoate are under evaluation [5–8]. It has been recently reported that bezafibrate stimulates FAO capacity by up-regulating MTP in human fibroblasts due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity [9]. Bezafibrate improved MTP deficiency in a subset of responsive genotypes. Since early recognition and prompt institution of therapy and appropriate preventive measures may be lifesaving and may significantly decrease long-term morbidity, genetic testing should be increasingly used. Here we report the spectrum of HADHA and HADHB variants that we detected by next generation sequencing in a cohort of 161 patients with clinical presentation of a myopathy and 242 patients with neuropathy and discuss the clinical phenotypes of molecularly confirmed diagnoses.
we applied a modified bioinformatic pipeline based on ExomeDepth 1.1.10, CANOES 2014, CLAMMS 1.0, and CODEX 1.5.0 (sensitivity 80.91%, PPV 95.62% at > 30-fold coverage). 2.3. Nomenclature and classification of genetic variants The nomenclature guidelines of the Human Genome Variation Society (HGVS) were used to describe the detected genetic variants [10]. The HADHA and HADHB missense variants were interpreted with the amino acid (AA) substitution prediction methods: Sorting Invariant from Tolerated (SIFT), PolyPhen-2, MutationTaster and MAPP. The Splice sites were predicted with: MES and SSF. The recurrence of the identified variants was established by interrogating the databases: Leiden Open Variation Database (LOVD), public archive of interpretations of clinically relevant variants (ClinVar) and the Human Gene Mutation Database (HGMD). The population databases: Database of all known Single Nucleotide Polymorphisms (dbSNP), Exome Aggregation Consortium (ExAC), Exome Sequencing Project (ESP) and Genome Aggregation Database (gnomAD) were used to determine the allele frequency of the variants. The variants were classified according to the ACMG guidelines with the 5-tier classification system: class 5 (pathogenic), class 4 (likely pathogenic), class 3 (variants of unknown significance, VUS), class 2 (likely benign) and class 1 (benign) [11]. 3. Results 3.1. Prevalence, classification and interpretation of HADHA and HADHB variants in a phenotype-based next generation sequencing cohort
2. Methods
The NGS data in our cohort of 403 patients with a phenotype of suspected metabolic myopathy (n = 161) or peripheral neuropathy (n = 242) were analyzed to identify MTP deficiency, and to assess the prevalence of MTP deficiency in this cohort. Filtering for variants of HADHA and HADHB, and classification of variants according to ACMG revealed nine different variants that were pathogenic or likely pathogenic (class 4 or 5 according to ACMG) in eleven independent individuals. Among them, ten (seven different) variants were found in HADHA, and two in HADHB (Table A.1). One patient was found to carry two heterozygous class 4/5 variants in HADHA (Patient ID 1, Table A.1). In total, HADHA sequencing identified seven heterozygous carriers of pathogenic variants (class 4/5). HADHB sequencing identified two heterozygous carriers of pathogenic variant (class 4/5). Additionally, we looked for a second variant in carriers of a class 4/5 variant in HADHA or HADHB gene. Interestingly, one patient carrying a class 4 variant in HADHA was identified to carry a second variant of uncertain significance (class 3) in a compound heterozygous state (Patient ID 2, Table A.1). Finally, we looked for patients with two class 3 variants in HADHA or HADHB gene and found one patient with a homozygous variant class 3 in HADHB (Patient ID 3, Table A.1). The remaining nine individuals (Patient ID 4–11, Table A.1) with single heterozygous class 4/5 variants in HADHA (n = 7) or HADHB (n = 2) did not reveal a second variant classified as class 3, 4, or 5 according to ACMG (Table A.1).
2.1. Subjects and study design To assess the prevalence of MTP deficiency and to verify the accuracy of phenotype-based variant interpretation, we re-analyzed data from next-generation sequencing (NGS) in a cohort of 403 patients with a phenotype that may mimic a late onset MTP deficiency. 161 patients presented with a leading clinical phenotype compatible with metabolic myopathy (recurrent hyper-CK-emia, muscle cramps, and/or myoglobinuria). 242 patients had a leading clinical phenotype of peripheral neuropathy. Peripheral blood samples (2–4 ml EDTA) or isolated DNA were received for each patient referred for molecular testing. Nextgeneration sequencing analysis was performed using a target panel that included more than 1500 genes associated with known human disease. For a targeted re-analysis of data, variants of HADHA (NM_000182) and HADHB (NM_000183) were filtered, and classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Segregation analysis to confirm compound heterozygosity of sequence variants in individual patients was done by Sanger sequencing in available family members. Informed consent was obtained from all participants and approved by local institutions (2018–213). 2.2. High throughput sequencing and bioinformatics pipeline Next-generation sequencing was carried out on an Illumina NextSeq 500 system (Illumina, San Diego, CA) as 150 bp paired-end runs using v2.0 SBS chemistry. Sequencing reads were aligned to the human reference genome (GRCh37/hg19) using BWA (v0.7.13-r1126) with standard parameters. Statistics on coverage and sequencing depth on the clinical targeted regions (i.e. RefSeq coding exons and ± 5 intronic region) were calculated with a custom script. SNV and INDEL calling on the nuclear genes was conducted using SAMtools (v1.3.1) with subsequent coverage and quality dependent filter steps. Variant annotation was performed with snpEff (v4.2) and Alamut-Batch (v1.4.4). Only variants (SNVs/small INDELs) in the coding and flanking intronic regions ( ± 50 bp) were evaluated. To identify heteroallelic copy number variations (CNVs) in the cohort of patients with heterozygous variants
3.2. Molecular diagnosis of HADHA/B-associated in three individuals of the cohort By NGS data analysis we identified three patients in a cohort of 403 patients with a peripheral neuropathy or metabolic myopathy phenotype. Patient 1 and 2 carried each two heterozygous variants in HADHA. Patient 3 carried a homozygous variant in HADHB (Table A.2). Patient 1 was identified to carry the heterozygous mutations c.180+3A > G, (p?) and c.1528G > C (p.Glu510Gln) in the HADHA gene (Table A.2 and Figure A.1). Both variants have been previously reported in a patient with recurrent rhabdomyolysis [12]. According to ACMG classification, they have to be classified as class 5 variants. The variant c.1528G > C (p.Glu510Gln) is the most common variant 15
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described in HADHA, which was observed in more than 90% of European patients with LCHAD/MTP deficiency [13,14]. The c.1528G > C substitution in exon 15, which alters glutamic acid into glutamine, within the catalytic region of the enzyme, results in LCHAD deficiency [15]. The variant is listed in population-specific databases dbSNP (rs137852769) and ClinVar (clinical significance: pathogenic).The variant c.180+3A > G, (p.?) affects the canonical 5´ splice site of exon 3, and is expected to cause abnormal gene splicing according to in silico splice prediction. Functional studies confirmed skipping of exon 3 resulting in undetectable levels of alpha subunit protein, and complete loss of trifunctional protein [16]. The variant is listed in populationspecific databases dbSNP (rs1057516417) and ClinVar (clinical significance:pathogenic). Compound heterozygosity is assumed but was not confirmed by analysis of parents. Patient 2 carried the heterozygous variants c.453+1G > T (p.?) and c.955G > A (p.Gly319Ser) in HADHA gene (Table A.2 and Figure A.1). Both mutations have not been reported previously in the literature. The variant c.453+1G > T (p.?) changes the canonical splice acceptor site of intron 5, and skipping of exon 5 is highly probable, even if not confirmed by functional studies. The variant is listed in population-specific databases dbSNP (rs1057516417) and ClinVar (clinical significance: likely pathogenic, class 4). The variant c.955G > A (p.Gly319Ser) is a non-conservative exonic variant predicted to result in the exchange of the highly conserved amino acid glycine in position 319 to serine with small physicochemical differences between glycine and serine (Grantham dist.: 56 0-215). [0–215]). The amino acid glycine at position 319 is in the “fatty acid oxidation complex, alpha subunit, mitochondrial” domain of the HADHA protein (Figure A.1) in a position that is conserved across species. In silico analysis predicts the resulting amino acid exchange to be probably damaging to the protein structure/function. The variant is listed in population-specific databases dbSNP (rs752317877), ExAC (8.242e-06) and gnomAD (0,0016%) and is listed in ClinVar RCV000624142.1 (Uncertain significance - Inborn genetic diseases), RCV000505768.1 (Uncertain significance not specified), RCV000665593.1 (Uncertain significance - Long-chain 3-hydroxyacylCoA dehydrogenase deficiency). We classified this variant also as a variant of uncertain significance (class 3). Compound heterozygosity of the two variants (c.955G > A (p.Gly319Ser), and c.453+1G > T (p.?)) in HADHA was confirmed by targeted parental testing. Patient 3 was identified to carry a homozygous variant in HADHB (NM_000183: c.712C > T (p.Arg238Trp) (Table A.2 and Figure A.2). The variant c.712C > T is listed in population-specific databases dbSNP (rs764006338), ExAC (8,239e-06) and gnomAD (0,008%) with a low frequency and is listed in ClinVar (RCV000318622.1, Uncertain significance – MTP deficiency). The variant results in a predicted amino acid exchange from arginine to tryptophan. The amino acids arginine and tryptophan show moderate physicochemical difference (Grantham dist.: 101 [0–215]). Arginine at position 238 is located in the thiolasedomain of the HADHB protein and is highly conserved, up to Baker's yeast (considering 12 species). Bioinformatic predictions (SIFT, Polyphen, Mutation Taster) suggest a pathogenic effect. According to the ACMG we classified c.712C > T (p.Arg238Trp) as a variant of uncertain significance (class 3). Carrier status of the parents was not confirmed.
levels were slightly elevated (up to 1.5x the upper limit of normal). Apart from a minimal increase of C12, acylcarnitine levels in plasma were normal. An ophthalmologic examination detected no abnormalities. Patient 2 – identified in the cohort of neuropathy patients - was found to carry two compound heterozygous variants [c.453+1G > T (p.?)] + [c.955G > A (p.Gly319Ser)] in the HADHA gene (Table A.2 and Figure A.1). The currently 18-year-old girl had a very interesting phenotype: the first manifestation suggested a metabolic myopathy, but in the course of the disease the clinical picture changed so that the phenotype of the patient was classified as hereditary neuropathy. The girl was the second child of non-consanguineous Italian parents, and was born at 40 weeks of gestation after an uneventful pregnancy. Newborn screening results were reported to be normal. She achieved first motor milestones on time, but muscular hypotonia was noted at the age of 6 months. Physiotherapy was initiated. She walked on tiptoes and fell frequently. Muscle weakness showed no day-time dependent fluctuations, but worsened each time she had an infection. The girl's cognitive development was normal. On clinical examination at the age of 4 years she had repeatedly normal blood acylcarnitine levels, however these were slightly increased during infections. Intermittently, increased serum creatine kinase (CK) levels (> 2x the upper limit of normal) were documented. Muscle biopsy at age of 6 years revealed unspecific findings including fiber type 2 predominance. There was no obvious lipid storage and no typical ragged red fibers. Electrocardiogram, Echocardiography and a brain MRI were normal. Taken together, the clinical findings suggested a metabolic myopathy. On clinical examination at age 9 she presented with exercise intolerance, moderate muscle weakness of all extremities, reduced lower extremity muscle volume (distal muscular atrophy), areflexia in lower limbs, foot-drop, and high-arched feet. Compared to age-matched peers, her walking distance was reduced to 50 meters (m). She had trouble climbing stairs, Food intake was hampered by a weakness of the masticatory muscles. Electrophysiological studies revealed an axonal sensorimotor polyneuropathy in lower and upper limbs. Patient 3 – identified in the cohort of neuropathy patients-was found to carry a homozygous variant c.712C > T (p.Arg238Trp) in HADHB (Table A.2 and Figure A.2). Patient 3 is a 13-year old boy of Turkish origin with neuropathy, sensorimotor deficits, muscle weakness, distal muscular atrophy and areflexia in lower limbs. His serum CK levels were significantly increased (> 10x the upper limit of normal) and he had two fever induced rhabdomyolysis. Acylcarnitines and VLCFA were normal, muscle biopsy showed non-specific findings: abnormal fibre size variations and fibre type1 predominance. 4. Discussion NGS analysis of 161 patients with metabolic myopathy and 242 patients with peripheral neuropathy revealed 11 variants in the HADHA or the HADHB genes. Disease-causing variants in HADHA were identified in a nine-year old girl with peripheral neuropathy, which was first misinterpreted as metabolic myopathy, and in a 17-year old young man with recurrent rhabdomyolysis, illustrating the highly variable clinical presentations of mutations in the HADHA gene. In addition, we found a homozygous missense variant in HADHB in a 13-year old boy with sensorimotor neuropathy, muscle weakness, and highly elevated CK levels. Several disease-causing variants have been identified in the HADHA or HADHB genes, and most of them affect the α-subunit. In a patient with recurrent rhabdomyolysis (patient 1), we identified the pathogenic variants [c.180+3A > G (p?)] and [c.1528G > C (p.Glu510Gln)] in the HADHA gene. This is in line with a previously published patient who carried the same variants and also manifested with adult-onset recurrent rhabdomyolysis [12]. Unlike our patient, he also showed signs of mild axonal peripheral neuropathy. Thus our data further indicate that MTP/LCHADdeficiency should be considered in patients with adult-onset recurrent rhabdomyolysis, especially in those with either clinically overt or subclinical peripheral neuropathy.
3.2.1. Clinical presentation of the three patients diagnosed with HADHA/Bassociated MTP deficiency Patient 1– identified in the cohort of myopathy patients-was found to carry two heterozygous variants [c.180+3A > G, (p?)] and [c.1528G > C (p.Glu510Gln)] in HADHA (Table A.2 und Figure A.1). Newborn screening of the patient from Germany was reported to be normal. With onset of 4–5 years of age the patient complained of episodes of muscle pain accompanied by tea-coloured urine. The 30–40 episodes reported were triggered by heavy exercise and acute and reversible myositis. The genetic diagnosis of a HADHA/B-associated MTP deficiency was established at the age of 17. Physical examination at that age was normal, there was no evidence of neuropathy or permanent myopathy. CK 16
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In a nine-year old girl with peripheral neuropathy (patient 2) we identified the compound heterozygous variants [c.453+1G > T (p.?)] + [c.955G > A (p.Gly319Ser)] in the HADHA gene. Whereas the splice variant c.453+1G > T is classified as likely pathogenic (class 4), the variant c.955G > A (p.Gly319Ser) is formally classified as a variant of uncertain significance (class 3). Since the amino acid glycine at position 319 is in the “fatty acid oxidation complex, alpha subunit, the mitochondrial” domain of the HADHA protein in a position that is conserved across species, the in silico analysis predicts this variant as probably damaging to the protein structure/function. Parental DNA analysis confirmed the heteroallelic combination of these two variants. Taking into consideration the detailed clinical data, the diagnosis of a HADHA-associated metabolic disorder is very likely, even though, further data, e.g. functional studies, are needed to formally classify c.955G > A (p.Gly319Ser) as pathogenic variant according to ACMG. In addition, in the absence of functional data we cannot conclude whether the biochemical phenotype would be that of an MTP multienzyme complex deficiency (combined LCHAD, LCEH, and LCKT deficiency) or whether the isolated LCHAD activity would be compromised by the HADHA missense variant. Finally, we identified a homozygous variant c.712C > T (p.Arg238Trp) in the HADHB gene in a 13-years old boy (patient 3) with sensori-motor neuropathy, distal muscle atrophy, and increased serum CK levels (> 10x the upper limit of normal). The variant has not been reported in the literature, it is listed in population-specific databases and has to be classified as a variant of uncertain significance (class 3) according to ACMG. Further data, e.g. functional studies are needed to definitely prove a biochemical relevance and pathogenicity of this variant. Nevertheless, the clinical phenotype of the patient makes an MTP deficiency very likely. In summary, our cases highlight the fact, that in some cases of a nonspecific myopathy, neuropathy, or a mixed myopathic-neuropathic disease the diagnosis of MTP/LCHAD deficiency can be established by NGS analysis and the interpretation of variants by ACMG classification guidelines, together with a depth clinical review of the patient's phenotype. In addition to the three patients carrying two heterozygous variants or a homozygous variant in one of the HADH genes, heterozygous carrier of pathogenic (class 5) and likely pathogenic (class 4) variants in the HADHA and HADHB gene were identified in our cohort (Table A.1, patient 4–11). In all these patients, we were not able to identify a second heteroallelic class 3, 4 or 5 variant or CNV, indicating a carrier status of an autosomal recessive disease. Of course, without further studies (genome sequencing, RNA sequencing) it cannot be excluded that a heteroallelic variant, e.g. an exonic/genomic rearrangement or a regulatory relevant variant lying deep intronic or far outside in noncoding regions is present but was not detected. The most common variant in HADHA, which we also found in one patient with myopathy is the LCHAD variant c.1528G > C (p.Glu510Gln) [13,14]. The carrier frequency in the overall population is 1:773 and shows great differences, with highest frequency in populations living near the Baltic sea [17]. The frequency of c.1528G > C carriers is 1:598 in European (non-Finnish), 1:256 in Finnish, in Latino 1:1222, in African 1:3562, in South Asian 1:6122 and zero in East Asian population (gnomAD v2.1) [17]. Since the carrier frequency of variants, in the HADHA and HADHB gene is high, both genes are included in the comprehensive Carrier Screening and Molecular Diagnostic Testing for Recessive Childhood Diseases [18], respectively. Timely diagnosis of affected individuals has several potential benefits, such as available and genetic counseling of patients and families about risks for relatives and in additional offspring. Importantly, heterozygous pregnant woman may rarely develop either acute fatty liver of pregnancy or hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome) when carrying a child with LCHADD [19]. Interestingly, Spiekerkoetter and coworkers reported two unrelated patients with lethal MTP deficiency with uniparental disomy (UPD) of chromosome 2 resulting in lethal trifunctional protein deficiency due to homozygous alpha-subunit mutations [20], indicating that MTP
deficiency is another disorder that has become manifest due to isodisomy of chromosome 2. An analysis of parental DNA for patient 3 was not possible. Therefore, we cannot exclude that in this non-consanguineous couple UPD of chromosome 2 is the underlying reason for the homozygous variant in the son. Since UPD may represent a common mechanism for very rare autosomal recessive diseases, confirmation of variants in parents should be considered whenever possible. Importantly, information of UPD greatly reduces the 25% risk normally used for recessive disorders. As in summary three patients were diagnosed with MTP/LCHAD deficiency in our cohort of over 400 patients, these data highlight that HADHA or HADHB-associated fatty acid oxidation disorders (FAODs) can be rarely found even after negative results of a newborn screening. Due to the variable clinical spectrum of the disease, it is not easy to differentiate isolated LCHAD from general MTP deficiency or from other FAODs [21–23]. A recently published study of Lotz and Coworkers reported a series of three patients with LCHAD/MTP deficiency, in whom diagnosis was missed by newborn screening, resulting in life-threatening metabolic decompensations within the first half year of life [24]. The three patients presented in our study had later-onset and less severe phenotypes and they were also not identified by newborn screening, further highlighting the importance of genetic testing for diagnosis in those patients. Overall, LCHAD/MTP deficiency exhibits substantial molecular, biochemical and clinical heterogeneity and no clear genotype-phenotype correlation [25,26]. Thus, diagnostic delays are common and clinicians need a high index of suspicion to recognize a metabolic disorder. The laboratory tests may be normal, particularly between symptomatic episodes and confirmatory testing for long-chain fatty acid disorders are challenging. Interpretation of genetic variants must be made with careful consideration of the individual clinical symptoms in each case. Especially as the pathogenicity of missense mutations in rare disease genes is due to a lack of data frequently not assigned and class 3 variants can only be assumed as disease causing in the light of the clinical phenotype and the compound heterozygote combination with other pathogenic sequence variants in the same gene. Our data confirm, that patients with milder, late-onset phenotypes of MTP deficiency, may present as metabolic myopathies or may even mimic hereditary neuropathy, highlighting the importance of genetic testing in regard of the clinical phenotype. In patients with suspected hereditary myopathy or neuropathy, phenotype-based NGS testing is now a frequently used diagnostic tool. The ACMG recommends that clinical sequencing laboratories return secondary findings in 56 genes associated with medically actionable conditions [27]. Although specific therapies are available for MTP/LCHAD deficiency, the HADHA and HADHB gene is not listed as actionable gene according to the ACMG guidelines so far. Diagnosis of MTP/LCHAD deficiency as a treatable metabolic disorder is highly relevant. Moreover, elucidating the underlying genetic defect might have impact on future molecular treatment options to progress the treatment of rare genetic disorders. 5. Conclusion Clinical symptoms of MTP deficiency vary between severe, early-onset, cardiac forms and milder, later-onset, myopathic and neuropathy phenotypes. Since newborn screening has limitations and clinical symptoms may be subtle, phenotype-based diagnostic via NGS should by increasingly used to identify the cause of MTP/LCHAD deficiency disorders. Disclosure statement The authors declare no conflict of interest. Acknowledgements We want to thank Kristina Lenhard for assistance with sample preparation and sequencing. 17
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Appendices Table A.1
HADHA and HADHB gene variants identified by phenotype-based NGS in cohort of 403 individuals ID
1 2
Individuals with two heterozygous variants (pathogenic or likely pathogenic)
Individuals with two heterozygous variants (one pathogenic or likely pathogenic; one VUS)
Individuals with two heterozygous or one Individuals with one heterozygous variant homozygous variants of unknown signifi- (pathogenic or likely pathogenic) cance
Allele 1 Class 4/5
Allele 2 Class 4/5
Allele 1 Class 4/5
Allele 2 Class 3
Allele 1 Class 3
Allele 2 Class 3
HADHA c.180+3A > G, (p.?)
HADHA c.1528G > C (p.Glu510Gln)
HADHA c.453+1G > T, (p.?)
HADHA c.955G > A (p.Gly319Ser)
HADHB c.712C > T (p.Arg238Trp)
HADHB c.712C > T (p.Arg238Trp)
3 4 5 6 7 8 9 10 11 12
Allele 1 Class 4/5
Allele 2 no call
HADHA c.859del (p.Glu287Lysfs*16) HADHA c.1561_1562del, p.Thr521Glnfs*19 HADHA c.2059del (p.Met687Cysfs*43) HADHA c.180_180+5delGGTATCinsAT (p.Val61Cysfs*6) HADHA c.1528G > C (p.Glu510Gln) HADHA c.1528G > C (p.Glu510Gln) HADHA c.1528G > C (p.Glu510Gln) HADHB c.631-1G > A (p?) HADHB c.580C > T:p.Gln194*
Phenotype-based Next Generation Sequencing (NGS) analysis were performed in a cohort of 403 individuals (161 patients with metabolic myopathy phenotype and 242 patients with peripheral neuropathy phenotype). HADHA and HADHB variants were classified according to ACMG (class 1 = benign, class 2 = likely benign, class 3 = variant of unknown significance, class 4 = likely pathogenic, class 5 = pathogenic). In total, filtering for variants of HADHA and HADHB revealed nine different variants that were pathogenic or likely pathogenic (class 4 or 5 according to ACMG) in twelve independent individuals. Ten variants were found in HADHA and two variants in HADHB. One patient was found to carry two heterozygous class 5 variants in HADHA (ID 1). Compound heterozygosity in patient 1 was not confirmed. HADHA sequencing identified seven heterozygous carriers of pathogenic variants (class 4/5) (ID 4–12). No second variant class 3, class 4 or class 5 was identified in those individuals (no call). HADHB sequencing identified two heterozygous carriers of pathogenic variant (class 4/5) (ID 11 + 12). One patient carrying a class 4 variant in HADHA was identified to carry a second variant of uncertain significance (class 3) in a compound heterozygous state (ID 2). One patient was found with a homozygous variant class 3 in HADHB (ID 3).
Table A.2
Clinical and genetic characteristics of three independent patients with variants in the HADHA or HADHB gene ID
age at diagnosis
allele 1
ACMG class
allele 2
ACMG class
Phenotype
1
17 y
HADHA
c. 180+3A > G (p.?)
5
5
Recurrent rhabdomyolyses, myoglobinuria
9y
HADHA
c. 453+1G > T (p.?)
4
3
13 y
HADHB
c. 712C > T (p.Arg238Trp)
3
c. 1528G > C (p.Glu510Gln) c. 955 > A (p.Gly319Ser) c.712C > T (p.Arg238Trp)
2
Neuropathy (NLG N. medianus > 38 m/s), distal muscle atrophy, exercice intolerance Neuropathy (axonal, sensorimotor), distal muscle atrophy, elevated creatine kinase (max. 2473 U/l), left rebnal dyplasia, small stature
3
3
Phenotype-based Next Generation Sequencing (NGS) diagnosed three patients by filtering for variants in HADHA or HADHB in cohort of 403 individuals with neuropathy or myopathy phenotype. HADHA and HADHB variants were classified according to ACMG (class 1 = benign, class 2 = likely benign, class 3 = variant of unknown significance, class 4 = likely pathogenic, class 5 = pathogenic). Patient 1 (ID 1) was identified in the cohort of myopathy patients. He was 18 years (y) at diagnosis. NGS analysis identified that patient 1 carries two compound heterozygous variants [c.180+3A > G (p?)] and [c.1528G > C (p.Glu510Gln)] in the HADHA (NM_000182.4). Compound heterozygosity in patient 1 was not confirmed. Patient 2 (ID 2) was identified in the cohort of neuropathy patients. Patient 2 was 9 y at age of diagnosis. Patient 2 was found to carry two compound heterozygous variants [c.453+1G > T (p.?)] + [c.955G > A (p.Gly319Ser)] in the HADHA. Compound heterozygosity in patient 2 was confirmed. Patient 3 (ID 3) was identified in the cohort of neuropathy patients. Patient 3 was found to carry a homozygous variant c.712C > T (p.Arg238Trp) in the HADHB (NM_000183.2). Compound heterozygosity in patient was confirmed.
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Fig. A.1. Domain structure of HADHA: Location of the identified variants in the HADHA protein: Domain structure of HADHA (Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha) with location of the variants in the HADHA protein is shown. The HADHA variants identified in patient 1 (ID 1, grey box) and patient 2 (ID 2, yellow box) were mapped to the known functional domains of the HADHA protein indicated in blue: (3HCDH: Hydroxyacyl-CoA-Dehydrogenase C-terminal Domain), green: (3HCDH_N: Hydroxyacyl-CoA-Dehydrogenase NAD-binding Domain), orange: (ECH Crotonase (EnoylCoA-Hydratase/Isomerase). Disease relevant HADHA mutations are indicated in grey boxes (ID 1) or yellow boxes (ID 2).
Fig. A 2. Domain structure of HADHB: Location of the identified variants in the HADHB protein: Domain structure of HADHB (Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Beta) with location of the variant in the HADHB protein is shown. The HADHB variant identified in patient 3 (ID 3, green box) was mapped to the known functional domains of the HADHB protein indicated in yellow: (Thiolase N), red: (Thiolase C). Disease relevant HADHB mutation is indicated in the green box.
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HADHA and HADHB gene associated phenotypes - Identification of rare variants in a patient cohort by Next Generation Sequencing
T
Isabel Diebolda, Ulrike Schöna, Rita Horvathc, Oliver Schwartzd, Elke Holinski-Federa, Heike Kölbele, Angela Abichta,b,∗ a
Medical Genetics Center, Munich, Germany Department of Neurology, Friedrich-Baur-Institute, Klinikum der Ludwig-Maximilians-University, Munich, Germany c Department of Clinical Neuroscience, University of Cambridge, Cambridge, UK d Department of Neuropediatrics, University Children's Hospital Muenster, Muenster, Germany e Department of Pediatric Neurology, Developmental Neurology and Social Pediatrics, University of Essen, Germany b
ARTICLE INFO
ABSTRACT
Keywords: Mitochondrial trifunctional protein HADHA HADHB Next generation sequencing Neuropathy Metabolic myopathy
The heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits harbours three enzymes that each perform a different function in mitochondrial fatty acid β-oxidation. Pathogenic variants in the MTP genes (HADHA and HADHB) cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by phenotypic heterogeneity ranging from severe, early-onset, cardiac disease to milder, later-onset, myopathy and neuropathy. Since metabolic myopathies and neuropathies are a group of rare genetic disorders and their associated muscle symptoms may be subtle, the diagnosis is often delayed. Here we evaluated data of 161 patients with myopathy and 242 patients with neuropathy via next generation sequencing (NGS) and report the diagnostic yield in three patients of this cohort by the detection of disease-causing variants in the HADHA or HADHB gene. The mitigated phenotypes of this treatable disease were missed by the newborn screening, highlighting the importance of phenotype-based NGS analysis in patients with rare and clinically very variable disorders such as MTP deficiency.
1. Introduction Mitochondrial trifunctional protein (MTP) is a large heteromultimeric enzyme that catalyzes the three final steps of long-chain mitochondrial fatty acid β-oxidation, providing an important energy source for skeletal muscle and the heart. MTP is composed of four hydroacyl-CoA dehydrogenase-α (HADHA) and four hydroacyl-CoA dehydrogenase-β (HADHB) subunits encoded by the HADHA and HADHB genes. The genes HADHA and HADHB are located in a head-to-head configuration on chromosome 2p23.3 and share a common promoter. The HADHA gene contains the information for the long-chain enoylCoA hydratase (LCEH) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), whereas the HADHB encodes long-chain ketoacylCoA thiolase (LCKT) activity. To date, two different biochemical phenotypes of defects in the MTP complex are known, the isolated LCHAD deficiency (LCHADD) (OMIM# 609016) and generalized MTP deficiency (OMIM# 609015). Both, isolated LCHAD and MTP deficiency, lead to an accumulation of toxic β-oxidation intermediates causing acute symptoms as well as long-term complications with particularly high morbidity and mortality. Clinical symptoms mainly
∗
develop during periods of illness or fasting and affect preferably the heart and the skeletal muscle. Thus, clinical manifestations of LCHAD/MTP deficiency can vary from early-onset cardiomyopathy, hypoglycemia, hepatopathy, coma, and sudden infant death syndrome (SIDS) to later onset myopathy, neuropathy and pigmentary retinopathy [1,2]. More than 60% of surviving individuals with LCHAD/MTP deficiency present with myopathy, about 20% develop a slowly progressing peripheral neuropathy, and more than 40% have pigmentary retinopathy [1]. Adults, adolescents or older children may develop recurrent rhabdomyolysis during illness. If LCHAD/MTP deficiency is diagnosed in time, outcome can be favorable, prompting the implementation in newborn screening programs [3]. Acylcarnitines are determined by tandem mass spectrometry with 3-hydroxypalmitoylcarnitine (C16eOH) and 3-hydroxyoleoylcarnitine (C18:1OH) as primary biomarkers [4]. However, there are limitations of newborn screening for LCHAD/MTP deficiency and clinical symptoms of children unidentified as newborns may be subtle. Deficiencies of MTP/LCHAD are autosomal recessively inherited disorders with an estimated frequency of 1:140,000. A prevalent missense mutation (c.1528G > C, (p.Glu510Gln)) in the LCHAD domain of the HADHA gene can be detected in approximately 90% of LCHAD-
Corresponding author. Medical Genetics Center, Munich, Germany. E-mail address: [email protected] (A. Abicht).
https://doi.org/10.1016/j.mcp.2019.01.003 Received 13 December 2018; Received in revised form 17 January 2019; Accepted 20 January 2019 Available online 22 January 2019 0890-8508/ © 2019 Elsevier Ltd. All rights reserved.
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deficient alleles, and patients homozygous for this variant show widely different phenotypes, thus making molecular screening for LCHADD important and feasible. Compared to LCHADD, the molecular basis of MTP deficiency is heterogeneous and different variants have been identified in both HADHA and HADHB genes with poor genotype–phenotype correlation. To date the Human Gene Mutation Database (HGMD) reports 64 HADHA and 60 HADHB gene mutations, many of them have not been functionally characterized. Treatment of LCHAD/MTP deficiency involves avoidance of prolonged fasting, dietary fat restriction, medium chain triglycerides (MCT) supplementation. A low-fat diet and medium chain triglycerides (MCT) supplementation decrease plasma hydroxyl acylcarnitine levels, and most patients remain healthy without metabolic decompensation but still require supplementation with essential fatty acids and fat-soluble vitamins [5,6]. Further treatment options such as anaplerotic therapy with heptanoate are under evaluation [5–8]. It has been recently reported that bezafibrate stimulates FAO capacity by up-regulating MTP in human fibroblasts due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity [9]. Bezafibrate improved MTP deficiency in a subset of responsive genotypes. Since early recognition and prompt institution of therapy and appropriate preventive measures may be lifesaving and may significantly decrease long-term morbidity, genetic testing should be increasingly used. Here we report the spectrum of HADHA and HADHB variants that we detected by next generation sequencing in a cohort of 161 patients with clinical presentation of a myopathy and 242 patients with neuropathy and discuss the clinical phenotypes of molecularly confirmed diagnoses.
we applied a modified bioinformatic pipeline based on ExomeDepth 1.1.10, CANOES 2014, CLAMMS 1.0, and CODEX 1.5.0 (sensitivity 80.91%, PPV 95.62% at > 30-fold coverage). 2.3. Nomenclature and classification of genetic variants The nomenclature guidelines of the Human Genome Variation Society (HGVS) were used to describe the detected genetic variants [10]. The HADHA and HADHB missense variants were interpreted with the amino acid (AA) substitution prediction methods: Sorting Invariant from Tolerated (SIFT), PolyPhen-2, MutationTaster and MAPP. The Splice sites were predicted with: MES and SSF. The recurrence of the identified variants was established by interrogating the databases: Leiden Open Variation Database (LOVD), public archive of interpretations of clinically relevant variants (ClinVar) and the Human Gene Mutation Database (HGMD). The population databases: Database of all known Single Nucleotide Polymorphisms (dbSNP), Exome Aggregation Consortium (ExAC), Exome Sequencing Project (ESP) and Genome Aggregation Database (gnomAD) were used to determine the allele frequency of the variants. The variants were classified according to the ACMG guidelines with the 5-tier classification system: class 5 (pathogenic), class 4 (likely pathogenic), class 3 (variants of unknown significance, VUS), class 2 (likely benign) and class 1 (benign) [11]. 3. Results 3.1. Prevalence, classification and interpretation of HADHA and HADHB variants in a phenotype-based next generation sequencing cohort
2. Methods
The NGS data in our cohort of 403 patients with a phenotype of suspected metabolic myopathy (n = 161) or peripheral neuropathy (n = 242) were analyzed to identify MTP deficiency, and to assess the prevalence of MTP deficiency in this cohort. Filtering for variants of HADHA and HADHB, and classification of variants according to ACMG revealed nine different variants that were pathogenic or likely pathogenic (class 4 or 5 according to ACMG) in eleven independent individuals. Among them, ten (seven different) variants were found in HADHA, and two in HADHB (Table A.1). One patient was found to carry two heterozygous class 4/5 variants in HADHA (Patient ID 1, Table A.1). In total, HADHA sequencing identified seven heterozygous carriers of pathogenic variants (class 4/5). HADHB sequencing identified two heterozygous carriers of pathogenic variant (class 4/5). Additionally, we looked for a second variant in carriers of a class 4/5 variant in HADHA or HADHB gene. Interestingly, one patient carrying a class 4 variant in HADHA was identified to carry a second variant of uncertain significance (class 3) in a compound heterozygous state (Patient ID 2, Table A.1). Finally, we looked for patients with two class 3 variants in HADHA or HADHB gene and found one patient with a homozygous variant class 3 in HADHB (Patient ID 3, Table A.1). The remaining nine individuals (Patient ID 4–11, Table A.1) with single heterozygous class 4/5 variants in HADHA (n = 7) or HADHB (n = 2) did not reveal a second variant classified as class 3, 4, or 5 according to ACMG (Table A.1).
2.1. Subjects and study design To assess the prevalence of MTP deficiency and to verify the accuracy of phenotype-based variant interpretation, we re-analyzed data from next-generation sequencing (NGS) in a cohort of 403 patients with a phenotype that may mimic a late onset MTP deficiency. 161 patients presented with a leading clinical phenotype compatible with metabolic myopathy (recurrent hyper-CK-emia, muscle cramps, and/or myoglobinuria). 242 patients had a leading clinical phenotype of peripheral neuropathy. Peripheral blood samples (2–4 ml EDTA) or isolated DNA were received for each patient referred for molecular testing. Nextgeneration sequencing analysis was performed using a target panel that included more than 1500 genes associated with known human disease. For a targeted re-analysis of data, variants of HADHA (NM_000182) and HADHB (NM_000183) were filtered, and classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Segregation analysis to confirm compound heterozygosity of sequence variants in individual patients was done by Sanger sequencing in available family members. Informed consent was obtained from all participants and approved by local institutions (2018–213). 2.2. High throughput sequencing and bioinformatics pipeline Next-generation sequencing was carried out on an Illumina NextSeq 500 system (Illumina, San Diego, CA) as 150 bp paired-end runs using v2.0 SBS chemistry. Sequencing reads were aligned to the human reference genome (GRCh37/hg19) using BWA (v0.7.13-r1126) with standard parameters. Statistics on coverage and sequencing depth on the clinical targeted regions (i.e. RefSeq coding exons and ± 5 intronic region) were calculated with a custom script. SNV and INDEL calling on the nuclear genes was conducted using SAMtools (v1.3.1) with subsequent coverage and quality dependent filter steps. Variant annotation was performed with snpEff (v4.2) and Alamut-Batch (v1.4.4). Only variants (SNVs/small INDELs) in the coding and flanking intronic regions ( ± 50 bp) were evaluated. To identify heteroallelic copy number variations (CNVs) in the cohort of patients with heterozygous variants
3.2. Molecular diagnosis of HADHA/B-associated in three individuals of the cohort By NGS data analysis we identified three patients in a cohort of 403 patients with a peripheral neuropathy or metabolic myopathy phenotype. Patient 1 and 2 carried each two heterozygous variants in HADHA. Patient 3 carried a homozygous variant in HADHB (Table A.2). Patient 1 was identified to carry the heterozygous mutations c.180+3A > G, (p?) and c.1528G > C (p.Glu510Gln) in the HADHA gene (Table A.2 and Figure A.1). Both variants have been previously reported in a patient with recurrent rhabdomyolysis [12]. According to ACMG classification, they have to be classified as class 5 variants. The variant c.1528G > C (p.Glu510Gln) is the most common variant 15
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described in HADHA, which was observed in more than 90% of European patients with LCHAD/MTP deficiency [13,14]. The c.1528G > C substitution in exon 15, which alters glutamic acid into glutamine, within the catalytic region of the enzyme, results in LCHAD deficiency [15]. The variant is listed in population-specific databases dbSNP (rs137852769) and ClinVar (clinical significance: pathogenic).The variant c.180+3A > G, (p.?) affects the canonical 5´ splice site of exon 3, and is expected to cause abnormal gene splicing according to in silico splice prediction. Functional studies confirmed skipping of exon 3 resulting in undetectable levels of alpha subunit protein, and complete loss of trifunctional protein [16]. The variant is listed in populationspecific databases dbSNP (rs1057516417) and ClinVar (clinical significance:pathogenic). Compound heterozygosity is assumed but was not confirmed by analysis of parents. Patient 2 carried the heterozygous variants c.453+1G > T (p.?) and c.955G > A (p.Gly319Ser) in HADHA gene (Table A.2 and Figure A.1). Both mutations have not been reported previously in the literature. The variant c.453+1G > T (p.?) changes the canonical splice acceptor site of intron 5, and skipping of exon 5 is highly probable, even if not confirmed by functional studies. The variant is listed in population-specific databases dbSNP (rs1057516417) and ClinVar (clinical significance: likely pathogenic, class 4). The variant c.955G > A (p.Gly319Ser) is a non-conservative exonic variant predicted to result in the exchange of the highly conserved amino acid glycine in position 319 to serine with small physicochemical differences between glycine and serine (Grantham dist.: 56 0-215). [0–215]). The amino acid glycine at position 319 is in the “fatty acid oxidation complex, alpha subunit, mitochondrial” domain of the HADHA protein (Figure A.1) in a position that is conserved across species. In silico analysis predicts the resulting amino acid exchange to be probably damaging to the protein structure/function. The variant is listed in population-specific databases dbSNP (rs752317877), ExAC (8.242e-06) and gnomAD (0,0016%) and is listed in ClinVar RCV000624142.1 (Uncertain significance - Inborn genetic diseases), RCV000505768.1 (Uncertain significance not specified), RCV000665593.1 (Uncertain significance - Long-chain 3-hydroxyacylCoA dehydrogenase deficiency). We classified this variant also as a variant of uncertain significance (class 3). Compound heterozygosity of the two variants (c.955G > A (p.Gly319Ser), and c.453+1G > T (p.?)) in HADHA was confirmed by targeted parental testing. Patient 3 was identified to carry a homozygous variant in HADHB (NM_000183: c.712C > T (p.Arg238Trp) (Table A.2 and Figure A.2). The variant c.712C > T is listed in population-specific databases dbSNP (rs764006338), ExAC (8,239e-06) and gnomAD (0,008%) with a low frequency and is listed in ClinVar (RCV000318622.1, Uncertain significance – MTP deficiency). The variant results in a predicted amino acid exchange from arginine to tryptophan. The amino acids arginine and tryptophan show moderate physicochemical difference (Grantham dist.: 101 [0–215]). Arginine at position 238 is located in the thiolasedomain of the HADHB protein and is highly conserved, up to Baker's yeast (considering 12 species). Bioinformatic predictions (SIFT, Polyphen, Mutation Taster) suggest a pathogenic effect. According to the ACMG we classified c.712C > T (p.Arg238Trp) as a variant of uncertain significance (class 3). Carrier status of the parents was not confirmed.
levels were slightly elevated (up to 1.5x the upper limit of normal). Apart from a minimal increase of C12, acylcarnitine levels in plasma were normal. An ophthalmologic examination detected no abnormalities. Patient 2 – identified in the cohort of neuropathy patients - was found to carry two compound heterozygous variants [c.453+1G > T (p.?)] + [c.955G > A (p.Gly319Ser)] in the HADHA gene (Table A.2 and Figure A.1). The currently 18-year-old girl had a very interesting phenotype: the first manifestation suggested a metabolic myopathy, but in the course of the disease the clinical picture changed so that the phenotype of the patient was classified as hereditary neuropathy. The girl was the second child of non-consanguineous Italian parents, and was born at 40 weeks of gestation after an uneventful pregnancy. Newborn screening results were reported to be normal. She achieved first motor milestones on time, but muscular hypotonia was noted at the age of 6 months. Physiotherapy was initiated. She walked on tiptoes and fell frequently. Muscle weakness showed no day-time dependent fluctuations, but worsened each time she had an infection. The girl's cognitive development was normal. On clinical examination at the age of 4 years she had repeatedly normal blood acylcarnitine levels, however these were slightly increased during infections. Intermittently, increased serum creatine kinase (CK) levels (> 2x the upper limit of normal) were documented. Muscle biopsy at age of 6 years revealed unspecific findings including fiber type 2 predominance. There was no obvious lipid storage and no typical ragged red fibers. Electrocardiogram, Echocardiography and a brain MRI were normal. Taken together, the clinical findings suggested a metabolic myopathy. On clinical examination at age 9 she presented with exercise intolerance, moderate muscle weakness of all extremities, reduced lower extremity muscle volume (distal muscular atrophy), areflexia in lower limbs, foot-drop, and high-arched feet. Compared to age-matched peers, her walking distance was reduced to 50 meters (m). She had trouble climbing stairs, Food intake was hampered by a weakness of the masticatory muscles. Electrophysiological studies revealed an axonal sensorimotor polyneuropathy in lower and upper limbs. Patient 3 – identified in the cohort of neuropathy patients-was found to carry a homozygous variant c.712C > T (p.Arg238Trp) in HADHB (Table A.2 and Figure A.2). Patient 3 is a 13-year old boy of Turkish origin with neuropathy, sensorimotor deficits, muscle weakness, distal muscular atrophy and areflexia in lower limbs. His serum CK levels were significantly increased (> 10x the upper limit of normal) and he had two fever induced rhabdomyolysis. Acylcarnitines and VLCFA were normal, muscle biopsy showed non-specific findings: abnormal fibre size variations and fibre type1 predominance. 4. Discussion NGS analysis of 161 patients with metabolic myopathy and 242 patients with peripheral neuropathy revealed 11 variants in the HADHA or the HADHB genes. Disease-causing variants in HADHA were identified in a nine-year old girl with peripheral neuropathy, which was first misinterpreted as metabolic myopathy, and in a 17-year old young man with recurrent rhabdomyolysis, illustrating the highly variable clinical presentations of mutations in the HADHA gene. In addition, we found a homozygous missense variant in HADHB in a 13-year old boy with sensorimotor neuropathy, muscle weakness, and highly elevated CK levels. Several disease-causing variants have been identified in the HADHA or HADHB genes, and most of them affect the α-subunit. In a patient with recurrent rhabdomyolysis (patient 1), we identified the pathogenic variants [c.180+3A > G (p?)] and [c.1528G > C (p.Glu510Gln)] in the HADHA gene. This is in line with a previously published patient who carried the same variants and also manifested with adult-onset recurrent rhabdomyolysis [12]. Unlike our patient, he also showed signs of mild axonal peripheral neuropathy. Thus our data further indicate that MTP/LCHADdeficiency should be considered in patients with adult-onset recurrent rhabdomyolysis, especially in those with either clinically overt or subclinical peripheral neuropathy.
3.2.1. Clinical presentation of the three patients diagnosed with HADHA/Bassociated MTP deficiency Patient 1– identified in the cohort of myopathy patients-was found to carry two heterozygous variants [c.180+3A > G, (p?)] and [c.1528G > C (p.Glu510Gln)] in HADHA (Table A.2 und Figure A.1). Newborn screening of the patient from Germany was reported to be normal. With onset of 4–5 years of age the patient complained of episodes of muscle pain accompanied by tea-coloured urine. The 30–40 episodes reported were triggered by heavy exercise and acute and reversible myositis. The genetic diagnosis of a HADHA/B-associated MTP deficiency was established at the age of 17. Physical examination at that age was normal, there was no evidence of neuropathy or permanent myopathy. CK 16
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In a nine-year old girl with peripheral neuropathy (patient 2) we identified the compound heterozygous variants [c.453+1G > T (p.?)] + [c.955G > A (p.Gly319Ser)] in the HADHA gene. Whereas the splice variant c.453+1G > T is classified as likely pathogenic (class 4), the variant c.955G > A (p.Gly319Ser) is formally classified as a variant of uncertain significance (class 3). Since the amino acid glycine at position 319 is in the “fatty acid oxidation complex, alpha subunit, the mitochondrial” domain of the HADHA protein in a position that is conserved across species, the in silico analysis predicts this variant as probably damaging to the protein structure/function. Parental DNA analysis confirmed the heteroallelic combination of these two variants. Taking into consideration the detailed clinical data, the diagnosis of a HADHA-associated metabolic disorder is very likely, even though, further data, e.g. functional studies, are needed to formally classify c.955G > A (p.Gly319Ser) as pathogenic variant according to ACMG. In addition, in the absence of functional data we cannot conclude whether the biochemical phenotype would be that of an MTP multienzyme complex deficiency (combined LCHAD, LCEH, and LCKT deficiency) or whether the isolated LCHAD activity would be compromised by the HADHA missense variant. Finally, we identified a homozygous variant c.712C > T (p.Arg238Trp) in the HADHB gene in a 13-years old boy (patient 3) with sensori-motor neuropathy, distal muscle atrophy, and increased serum CK levels (> 10x the upper limit of normal). The variant has not been reported in the literature, it is listed in population-specific databases and has to be classified as a variant of uncertain significance (class 3) according to ACMG. Further data, e.g. functional studies are needed to definitely prove a biochemical relevance and pathogenicity of this variant. Nevertheless, the clinical phenotype of the patient makes an MTP deficiency very likely. In summary, our cases highlight the fact, that in some cases of a nonspecific myopathy, neuropathy, or a mixed myopathic-neuropathic disease the diagnosis of MTP/LCHAD deficiency can be established by NGS analysis and the interpretation of variants by ACMG classification guidelines, together with a depth clinical review of the patient's phenotype. In addition to the three patients carrying two heterozygous variants or a homozygous variant in one of the HADH genes, heterozygous carrier of pathogenic (class 5) and likely pathogenic (class 4) variants in the HADHA and HADHB gene were identified in our cohort (Table A.1, patient 4–11). In all these patients, we were not able to identify a second heteroallelic class 3, 4 or 5 variant or CNV, indicating a carrier status of an autosomal recessive disease. Of course, without further studies (genome sequencing, RNA sequencing) it cannot be excluded that a heteroallelic variant, e.g. an exonic/genomic rearrangement or a regulatory relevant variant lying deep intronic or far outside in noncoding regions is present but was not detected. The most common variant in HADHA, which we also found in one patient with myopathy is the LCHAD variant c.1528G > C (p.Glu510Gln) [13,14]. The carrier frequency in the overall population is 1:773 and shows great differences, with highest frequency in populations living near the Baltic sea [17]. The frequency of c.1528G > C carriers is 1:598 in European (non-Finnish), 1:256 in Finnish, in Latino 1:1222, in African 1:3562, in South Asian 1:6122 and zero in East Asian population (gnomAD v2.1) [17]. Since the carrier frequency of variants, in the HADHA and HADHB gene is high, both genes are included in the comprehensive Carrier Screening and Molecular Diagnostic Testing for Recessive Childhood Diseases [18], respectively. Timely diagnosis of affected individuals has several potential benefits, such as available and genetic counseling of patients and families about risks for relatives and in additional offspring. Importantly, heterozygous pregnant woman may rarely develop either acute fatty liver of pregnancy or hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome) when carrying a child with LCHADD [19]. Interestingly, Spiekerkoetter and coworkers reported two unrelated patients with lethal MTP deficiency with uniparental disomy (UPD) of chromosome 2 resulting in lethal trifunctional protein deficiency due to homozygous alpha-subunit mutations [20], indicating that MTP
deficiency is another disorder that has become manifest due to isodisomy of chromosome 2. An analysis of parental DNA for patient 3 was not possible. Therefore, we cannot exclude that in this non-consanguineous couple UPD of chromosome 2 is the underlying reason for the homozygous variant in the son. Since UPD may represent a common mechanism for very rare autosomal recessive diseases, confirmation of variants in parents should be considered whenever possible. Importantly, information of UPD greatly reduces the 25% risk normally used for recessive disorders. As in summary three patients were diagnosed with MTP/LCHAD deficiency in our cohort of over 400 patients, these data highlight that HADHA or HADHB-associated fatty acid oxidation disorders (FAODs) can be rarely found even after negative results of a newborn screening. Due to the variable clinical spectrum of the disease, it is not easy to differentiate isolated LCHAD from general MTP deficiency or from other FAODs [21–23]. A recently published study of Lotz and Coworkers reported a series of three patients with LCHAD/MTP deficiency, in whom diagnosis was missed by newborn screening, resulting in life-threatening metabolic decompensations within the first half year of life [24]. The three patients presented in our study had later-onset and less severe phenotypes and they were also not identified by newborn screening, further highlighting the importance of genetic testing for diagnosis in those patients. Overall, LCHAD/MTP deficiency exhibits substantial molecular, biochemical and clinical heterogeneity and no clear genotype-phenotype correlation [25,26]. Thus, diagnostic delays are common and clinicians need a high index of suspicion to recognize a metabolic disorder. The laboratory tests may be normal, particularly between symptomatic episodes and confirmatory testing for long-chain fatty acid disorders are challenging. Interpretation of genetic variants must be made with careful consideration of the individual clinical symptoms in each case. Especially as the pathogenicity of missense mutations in rare disease genes is due to a lack of data frequently not assigned and class 3 variants can only be assumed as disease causing in the light of the clinical phenotype and the compound heterozygote combination with other pathogenic sequence variants in the same gene. Our data confirm, that patients with milder, late-onset phenotypes of MTP deficiency, may present as metabolic myopathies or may even mimic hereditary neuropathy, highlighting the importance of genetic testing in regard of the clinical phenotype. In patients with suspected hereditary myopathy or neuropathy, phenotype-based NGS testing is now a frequently used diagnostic tool. The ACMG recommends that clinical sequencing laboratories return secondary findings in 56 genes associated with medically actionable conditions [27]. Although specific therapies are available for MTP/LCHAD deficiency, the HADHA and HADHB gene is not listed as actionable gene according to the ACMG guidelines so far. Diagnosis of MTP/LCHAD deficiency as a treatable metabolic disorder is highly relevant. Moreover, elucidating the underlying genetic defect might have impact on future molecular treatment options to progress the treatment of rare genetic disorders. 5. Conclusion Clinical symptoms of MTP deficiency vary between severe, early-onset, cardiac forms and milder, later-onset, myopathic and neuropathy phenotypes. Since newborn screening has limitations and clinical symptoms may be subtle, phenotype-based diagnostic via NGS should by increasingly used to identify the cause of MTP/LCHAD deficiency disorders. Disclosure statement The authors declare no conflict of interest. Acknowledgements We want to thank Kristina Lenhard for assistance with sample preparation and sequencing. 17
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Appendices Table A.1
HADHA and HADHB gene variants identified by phenotype-based NGS in cohort of 403 individuals ID
1 2
Individuals with two heterozygous variants (pathogenic or likely pathogenic)
Individuals with two heterozygous variants (one pathogenic or likely pathogenic; one VUS)
Individuals with two heterozygous or one Individuals with one heterozygous variant homozygous variants of unknown signifi- (pathogenic or likely pathogenic) cance
Allele 1 Class 4/5
Allele 2 Class 4/5
Allele 1 Class 4/5
Allele 2 Class 3
Allele 1 Class 3
Allele 2 Class 3
HADHA c.180+3A > G, (p.?)
HADHA c.1528G > C (p.Glu510Gln)
HADHA c.453+1G > T, (p.?)
HADHA c.955G > A (p.Gly319Ser)
HADHB c.712C > T (p.Arg238Trp)
HADHB c.712C > T (p.Arg238Trp)
3 4 5 6 7 8 9 10 11 12
Allele 1 Class 4/5
Allele 2 no call
HADHA c.859del (p.Glu287Lysfs*16) HADHA c.1561_1562del, p.Thr521Glnfs*19 HADHA c.2059del (p.Met687Cysfs*43) HADHA c.180_180+5delGGTATCinsAT (p.Val61Cysfs*6) HADHA c.1528G > C (p.Glu510Gln) HADHA c.1528G > C (p.Glu510Gln) HADHA c.1528G > C (p.Glu510Gln) HADHB c.631-1G > A (p?) HADHB c.580C > T:p.Gln194*
Phenotype-based Next Generation Sequencing (NGS) analysis were performed in a cohort of 403 individuals (161 patients with metabolic myopathy phenotype and 242 patients with peripheral neuropathy phenotype). HADHA and HADHB variants were classified according to ACMG (class 1 = benign, class 2 = likely benign, class 3 = variant of unknown significance, class 4 = likely pathogenic, class 5 = pathogenic). In total, filtering for variants of HADHA and HADHB revealed nine different variants that were pathogenic or likely pathogenic (class 4 or 5 according to ACMG) in twelve independent individuals. Ten variants were found in HADHA and two variants in HADHB. One patient was found to carry two heterozygous class 5 variants in HADHA (ID 1). Compound heterozygosity in patient 1 was not confirmed. HADHA sequencing identified seven heterozygous carriers of pathogenic variants (class 4/5) (ID 4–12). No second variant class 3, class 4 or class 5 was identified in those individuals (no call). HADHB sequencing identified two heterozygous carriers of pathogenic variant (class 4/5) (ID 11 + 12). One patient carrying a class 4 variant in HADHA was identified to carry a second variant of uncertain significance (class 3) in a compound heterozygous state (ID 2). One patient was found with a homozygous variant class 3 in HADHB (ID 3).
Table A.2
Clinical and genetic characteristics of three independent patients with variants in the HADHA or HADHB gene ID
age at diagnosis
allele 1
ACMG class
allele 2
ACMG class
Phenotype
1
17 y
HADHA
c. 180+3A > G (p.?)
5
5
Recurrent rhabdomyolyses, myoglobinuria
9y
HADHA
c. 453+1G > T (p.?)
4
3
13 y
HADHB
c. 712C > T (p.Arg238Trp)
3
c. 1528G > C (p.Glu510Gln) c. 955 > A (p.Gly319Ser) c.712C > T (p.Arg238Trp)
2
Neuropathy (NLG N. medianus > 38 m/s), distal muscle atrophy, exercice intolerance Neuropathy (axonal, sensorimotor), distal muscle atrophy, elevated creatine kinase (max. 2473 U/l), left rebnal dyplasia, small stature
3
3
Phenotype-based Next Generation Sequencing (NGS) diagnosed three patients by filtering for variants in HADHA or HADHB in cohort of 403 individuals with neuropathy or myopathy phenotype. HADHA and HADHB variants were classified according to ACMG (class 1 = benign, class 2 = likely benign, class 3 = variant of unknown significance, class 4 = likely pathogenic, class 5 = pathogenic). Patient 1 (ID 1) was identified in the cohort of myopathy patients. He was 18 years (y) at diagnosis. NGS analysis identified that patient 1 carries two compound heterozygous variants [c.180+3A > G (p?)] and [c.1528G > C (p.Glu510Gln)] in the HADHA (NM_000182.4). Compound heterozygosity in patient 1 was not confirmed. Patient 2 (ID 2) was identified in the cohort of neuropathy patients. Patient 2 was 9 y at age of diagnosis. Patient 2 was found to carry two compound heterozygous variants [c.453+1G > T (p.?)] + [c.955G > A (p.Gly319Ser)] in the HADHA. Compound heterozygosity in patient 2 was confirmed. Patient 3 (ID 3) was identified in the cohort of neuropathy patients. Patient 3 was found to carry a homozygous variant c.712C > T (p.Arg238Trp) in the HADHB (NM_000183.2). Compound heterozygosity in patient was confirmed.
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Fig. A.1. Domain structure of HADHA: Location of the identified variants in the HADHA protein: Domain structure of HADHA (Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha) with location of the variants in the HADHA protein is shown. The HADHA variants identified in patient 1 (ID 1, grey box) and patient 2 (ID 2, yellow box) were mapped to the known functional domains of the HADHA protein indicated in blue: (3HCDH: Hydroxyacyl-CoA-Dehydrogenase C-terminal Domain), green: (3HCDH_N: Hydroxyacyl-CoA-Dehydrogenase NAD-binding Domain), orange: (ECH Crotonase (EnoylCoA-Hydratase/Isomerase). Disease relevant HADHA mutations are indicated in grey boxes (ID 1) or yellow boxes (ID 2).
Fig. A 2. Domain structure of HADHB: Location of the identified variants in the HADHB protein: Domain structure of HADHB (Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Beta) with location of the variant in the HADHB protein is shown. The HADHB variant identified in patient 3 (ID 3, green box) was mapped to the known functional domains of the HADHB protein indicated in yellow: (Thiolase N), red: (Thiolase C). Disease relevant HADHB mutation is indicated in the green box.
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