The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line.
The introduction of whole exome sequencing (WES) in clinical settings has massively augmented diagnostic yield for intellectual disability (ID) and other neurodevelopmental disorders (NDD), and additionally identified many new disease-gene associations. Yet, ~50–70% of individuals with ID/NDD remain undiagnosed . The Solve-RD project  systematically reanalyzes exomes and phenotypic data of 19,000 unsolved cases with rare disease from four European Reference Networks (ERNs) to elucidate the genetic etiology, including ~5,000 cases from ERN-ITHACA (Intellectual Disability, TeleHealth and Congenital Anomalies; https://ern-ithaca.eu/). Exploration of mitochondrial DNA sequences extracted from WES data is part of this effort , as 27 of the 37 mitochondrial genes have a known disease-gene association (http://www.mitomap.org).
MT-TL1 encodes mitochondrial tRNALeu(UUR), involved in the synthesis of oxidative phosphorylation enzymes by adding leucine to the growing polypeptide chain of mtDNA-encoded subunits during translation . Pathogenic variants in MT-TL1 have been linked to several phenotypes associated with mitochondrial dysfunction , including mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS; MIM#540000) and myoclonic epilepsy associated with ragged-red fibers (MERRF; MIM#545000).
We report on a variant in MT-TL1 known to interfere with mitochondrial function, uncovered by systematic re-analysis of WES data, illustrating the underexposed potential of WES-based analysis of mtDNA in identifying variants with clinical consequences.
All individuals (or legal representatives) in the Solve-RD project provided consent, compliant with local ethical guidelines and the Declaration of Helsinki. For this case, the Radboudumc Ethics Board approved the study (2018-4986). For publication of photos, additional consent was obtained.
Diagnostic trio-based exome sequencing (proband and parents) was performed as described previously  using DNA isolated from whole blood.
Human Phenotype Ontology-coded clinical data were uploaded along with BAM files to the RD-Connect Genome-Phenome Analysis Platform (https://platform.rd-connect.eu/), and deposited at European Genome-Phenome Archive (EGAZ00001527897), as part of the Solve-RD infrastructure . The variant and phenotype were submitted to the Leiden Open Variation Database (individual number 00328346, phenotype number 0000246573, variant number 0000713909).
Systematic re-analysis of WES data is described by Matalonga et al. . Details specific to this case include mapping, calling, and annotation of mtDNA using MToolBox pipeline (version 1.0) , with mapping against rCRS (for mtDNA) and GRCh37/Hg19 (for genomic DNA) as reference sequences, allowing the detection of heteroplasmy levels and prioritization of variants. The following parameters were applied to identify possible disease-associated variants: (1) coverage ≥8-fold; (2) heteroplasmy fraction ≥1%; (3) GeneBank allele frequency (MITOMAP) <0.2%; (4) “Confirmed” or “Reported” disease association in MITOMAP; and (5) reported “Pathogenic” (ACMG, class 5) or “Likely pathogenic” (ACMG, class 4) in ClinVar.
Confirmation of mitochondrial heteroplasmy was performed on blood and urine of the proband, mother and sisters using routine diagnostic procedures (PGM Ion Torrent Technology).
We report on a 23-year-old male proband with a complex neurodevelopmental and neuromuscular phenotype, who remained undiagnosed despite extensive diagnostic evaluation in a tertiary center. Family history was unremarkable, with two healthy older sisters and non-consanguineous parents. After uncomplicated pregnancy and delivery at term (normal birth weight (3840 g) and length (52 cm); head circumference 34 cm, 0 SD; Apgar score 10), first concerns about development arose around 3 months of age. At age 15 months, there was severe developmental delay, consisting of hypotonia, delayed motor, social and communicative milestones, and secondary microcephaly (44 cm, −2.5 SD). Brain MRI (at 15 months; repeated at age 14 years) showed supratentorial pachygyria and frontoparietal polymicrogyria (Fig. 1A), with white matter abnormalities in the posterior limb of the internal capsule (Fig. 1B). Cerebellum and corpus callosum showed no deformities and EEG did not show epileptiform activity at age 15 months.
He developed spastic tetraparesis, orofacial dystonia, and dystonia of hands and feet. Epilepsy manifested at 7 years of age with frontal focal-onset seizures, with and without secondary tonic-clonic generalization. No myoclonus was observed. Deterioration occurred at age 17 with episodes of severe headaches, accompanied by nausea, vomiting, hematemesis, pallor and perspiration, coinciding with epileptiform activity on EEG in the left temporal lobe. Brain CT was interpreted as normal at that time (Fig. 1C). Different anti-epileptic drugs were prescribed, of which several resulted in adverse drug reactions including erythema multiforme (carbamazepine, oxcarbazepine), muscle weakness (clobazam, pregabalin), or obstructive sleep apnea syndrome (clobazam), resolving after cessation of the respective medication. Because of poor seizure control, 24-h continuous EEG monitoring was performed at 18 years and showed bilateral independent and simultaneous periodic discharges, which were more frequent during sleep than during wakefulness. He is now largely seizure free on low-dose carbamazepine. Other medical problems were severe progressive neuromuscular scoliosis (Fig. 2A), bilateral hip dysplasia with luxation, drooling with excessive tenacious stringy mucus, and severe feeding difficulties requiring gastric tube feeding and resulting in low body weight (age 18: length 165 cm, −2.6 SD; weight 39.5 kg, weight-for-length −3.1 SD; head circumference 54 cm, −1.9 SD). Ophthalmological assessment, visual and brainstem auditory evoked potentials, and electrocardiography were normal. Facial dysmorphisms at 19 years of age included a long hypotonic face, full eyebrows, long palpebral fissures, a prominent nose and nasal bridge, high palate, gingival hyperplasia, and abnormal tooth implant (Fig. 2B–D), becoming more prominent over time (Fig. 2E).
Karyotype, genomic micro-array (Agilent 180k oligo-array), targeted analysis of WDR62 and ADGRG1, extensive lysosomal screening, and trio-based WES did not yield a diagnosis. Metabolic workup revealed elevation of multiple amino acids, including glycine, serine, threonine (in plasma and urine), lysine, methionine, and alanine (in plasma), in a pattern not described for a known metabolic disorder. Lactate in blood (1.8 mmol/l, age 20 years) and cerebrospinal fluid (1.0 mmol/l, age 15 months) were within normal range. Pharmacogenetic analysis showed a rare homozygous variant in CYP3A4 (NM_017460.5:c.878T > C;*18; rs28371759) unlikely to explain the largely dose-independent adverse drug reactions, and two rare HLA-types (HLA-A*0103; HLA-B*0835) of unknown significance.
Systematic re-analysis revealed a known pathogenic variant in MT-TL1
Data of the proband and parents were included in the Solve-RD project. Prioritization of nuclear DNA variants did not yield diagnostically relevant variants, despite analysis of (de novo) variants in known disease–genes, particularly those associated with recessive or dominant cortical dysplasia, and in genes not yet implicated in NDD/ID. Variant prioritization from mtDNA revealed a variant in MT-TL1 , NC_012920.1:m.3291T > C (NC_012920.1:n.62T > C), known to affect mitochondrial function, at 22% heteroplasmy. The variant was absent from maternal WES data.
Validation was performed on blood of the proband and his mother using routine diagnostic procedures, displaying a heteroplasmy level of 23% in the index, compared to 4% in his mother. Urine heteroplasmy levels were 58% and 17% in index and mother, respectively. Follow-up of the family revealed heteroplasmy levels of 4% (blood) and 9% (urine) in one of the sisters, but the variant was absent from blood and urine of the other sister. We re-evaluated brain CT imaging of the index, performed at age 17 years, which in retrospect showed early signs of stroke, including loss of gray–white differentiation in the left temporal lobe (Fig. 1C), co-localizing with epileptiform activity seen on EEG at the time of onset of episodic symptoms. Additionally, multidisciplinary evaluation and comparison of his phenotype to previously published individuals with the same variant (Table 1) revealed that the proband exhibits several symptoms seen in other individuals (epilepsy, episodic headaches with emesis, feeding difficulties, low body weight, and neuromuscular problems) [9,10,11,12,13,14], but also features that were not described before (abnormalities of brain gyration, facial dysmorphisms, early age of onset of developmental delay).
Systematic re-analysis of existing WES data of unresolved cohorts can efficiently yield additional diagnoses . Yet, re-analysis rarely includes evaluation of mtDNA although pathogenic mtDNA variants underly many rare diseases. This case illustrates the importance of including mtDNA in re-analysis, as the identification of the MT-TL1 variant is of medical relevance to the proband and his sisters of childbearing age.
Mitochondrial disorders associated with mitochondrial tRNA genes are characterized by both genotypic and phenotypic heterogeneity, with poor genotype–phenotype correlations [8, 16, 17]. The m.3291T > C variant described here is located in the T-loop of mitochondrial tRNALeu(UUR). This variant was shown to result in respiratory chain enzyme deficiency and its pathogenicity was proven by single muscle fiber mtDNA analysis, showing high heteroplasmy levels in cytochrome c oxidase deficient muscle fibers . Individuals carrying the m.3291T > C variant display a broad phenotypic spectrum varying from mild symptoms to severely debilitating disease [8,9,10,11,12,13,14, 18, 19], largely overlapping with features observed in the index. As no additional diagnostically relevant variants could be identified, it remains unclear whether the early age of onset of developmental delay, gyration defects, and dysmorphisms are attributable to a second rare genetic disorder, or expand the m.3291T > C phenotypic spectrum. However, despite gyration defects being uncommon in individuals with MT-TL1 variants, polymicrogyria has been reported for m.3243A > G in two unrelated individuals with atypical MELAS phenotypes, additionally exhibiting other atypical symptoms also observed in the proband, including hypertonia, early onset developmental delay [20, 21] and facial dysmorphisms . The proband’s facial dysmorphisms might be secondary to muscle tone abnormalities. Hence, we concluded that the MT-TL1 variant could at least in part, but possibly completely, explain the proband’s phenotype.
In conclusion, we describe a male proband carrying a mtDNA variant confirmed to interfere with mitochondrial function, that was identified in systematic, large-scale re-analysis on a large cohort of individuals with unresolved (syndromic) ID through the collaborative Solve-RD project. Our observations suggest that re-analysis encompassing the mtDNA interpreted from WES data may successfully yield novel unanticipated diagnoses in unexplained cases of ERN-ITHACA with implications for reproductive choices of relatives in the maternal line.
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We are extremely grateful to the proband and his parents for their willingness to participate in this study. In addition, we thank Michael Kwint, Kornelia Neveling, Fons Stassen, Steve Laurie, and Raul Tonda for their technical and bioinformatic support. This work has been generated through a collaboration with the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability (ERN-ITHACA) [EU Framework Partnership Agreement ID: 3HP-HP-FPA ERN-01-2016/739516]. For more information about the ERNs and the EU health strategy please visit http://ec.europa.eu/health/ern.
This work was financially supported by Aspasia grants of the Dutch Research Council (015.014.036 to TK and 015.014.066 to LELMV), the European Research Council (ERC to RH), the Wellcome Investigator Award (109915/Z/15/Z to RH), the Medical Research Council UK (MR/N025431/1 to RH), the Newton Fund (MR/N027302/1 to RH), the Lily Foundation (RH), and the Evelyn Trust (RH). The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 779257.
Conflict of interest
The authors declare no competing interests.
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Members of the Solve-RD SNV-indel working group and Solve-RD-DITF-ITHACA are listed in Supplementary Information.
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de Boer, E., Ockeloen, C.W., Matalonga, L. et al. A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis. Eur J Hum Genet (2021). https://doi.org/10.1038/s41431-021-00900-2