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A recessive variant in TFAM causes mtDNA depletion associated with primary ovarian insufficiency, seizures, intellectual disability and hearing loss

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Mitochondrial disorders are collectively common, genetically heterogeneous disorders in both pediatric and adult populations. They are caused by molecular defects in oxidative phosphorylation, failure of essential bioenergetic supply to mitochondria, and apoptosis. Here, we present three affected individuals from a consanguineous family of Pakistani origin with variable seizures and intellectual disability. Both females display primary ovarian insufficiency (POI), while the male shows abnormal sex hormone levels. We performed whole exome sequencing and identified a recessive missense variant c.694C > T, p.Arg232Cys in TFAM that segregates with disease. TFAM (mitochondrial transcription factor A) is a component of the mitochondrial replisome machinery that maintains mtDNA transcription and replication. In primary dermal fibroblasts, we show depletion of mtDNA and significantly altered mitochondrial function and morphology. Moreover, we observed reduced nucleoid numbers with significant changes in nucleoid size or shape in fibroblasts from an affected individual compared to controls. We also investigated the effect of tfam impairment in zebrafish; homozygous tfam mutants carrying an in-frame c.141_149 deletion recapitulate the mtDNA depletion and ovarian dysgenesis phenotypes observed in affected humans. Together, our genetic and functional data confirm that TFAM plays a pivotal role in gonad development and expands the repertoire of mitochondrial disease phenotypes.

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Availability of data and material

Data, reagents, and mutant animal lines are available upon request and contingent upon appropriate ethics protocols from the requesting investigator and institution.

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Not applicable. Publicly available software was used for data analysis.


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We are grateful to all the members of the families reported in this study for their participation and support of our research. We thank Azita Sadeghpour for facilitating primary cell line establishment. We thank the Beijing Genomics Institute (BGI) staff for technical support in generating WES data. We thank Niki Mourtzi for generating jointly called vcf files and Marie Mooney for critical review of the manuscript.


This study was funded by the Higher Education Commission of Pakistan under the International research support initiative program (IRSIP program; to FU); an Australian National Health and Medical Research Council (NHMRC) program grant (1074258, AHS); an Australian Mito Foundation grant (EJT); NHMRC fellowships (1054432 EJT, 1062854 AHS); and the United States National Institutes of Health grant P50-HD028138 (EED). EED is the Ann Marie and Francis Klocke MD Research Scholar.

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Authors and Affiliations



FU, WR, NK, SB and EED contributed to the conception and design of the study. SK and MT performed clinical investigation and ascertainment of the biological specimens for family 1. SH conducted WES and bioinformatic filtering. FU and KK validated WES data from family 1. FU and KK performed in vitro studies of primary cells. FU generated and phenotyped zebrafish models. KB, VCO, TP, AS and ET contributed data from family 2. KMB performed IBD analysis. FU, KK and EED drafted the manuscript. All authors read the manuscript and approved the submitted version.

Corresponding author

Correspondence to Erica E. Davis.

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Conflicts of interest

NK is a significant shareholder in Rescindo Therapeutics.

Ethics approval

Human subjects research was approved by the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, and Lurie Children’s’ Hospital of Chicago, IL, USA. All zebrafish experiments were approved by institutional animal care and use committees (IACUC) of Duke University and Northwestern University.

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Written informed consent was obtained from all willing research participants.

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Written informed consent was obtained from all willing research participants for publication.

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Supplementary file1 (DOCX 32 kb)


Figure S1. Homozygosity mapping shows a shared region between affected individuals in family 1. (A) Homozygous regions were mapped in affected individuals using HomozygosityMapper software. The homozygous blocks (red) were identified on chromosomes 6, 8 and 10 with >80% homozygosity. (B) Schematic representation of chromosome 10. TFAM is located on 10q21.2 (blue box). (C) Enlarged view of the chromosome 10 region shared between affected individuals (1-V-I and 1-V-II). (D) High resolution graphical representation of the homozygous region on chromosome 10 generated with GeneDistiller


Figure S2. In silico protein modeling of TFAM p.Arg232Cys. Human TFAM protein sequence harboring either Arg232 (WT; top) or Cys232 (variant; bottom) was modeled in PyMOL and depicts TFAM (green) co-crystalized in a complex with light strand promoter (LSP; grey) sequence. The zoomed image (top right) shows Arg232 (cyan) interacting with Glu219 (red). Arginine changed to cysteine at position 232 may impair the interaction with glutamic acid at position 219 (bottom right) by disrupting the salt bridge present between these two amino acids in WT protein


Figure S3. CRISPR-Cas9 genome editing was used to target tfam in zebrafish. (A) Schematic shows the canonical transcript of tfam (GRCz11, ENSDART00000092009.6), green boxes, coding exons; untranslated regions (UTR), white boxes; black lines, introns. Exon targeted by sgRNA is shown with a black arrow. (B) Polyacrylamide gel image showing heteroduplex analysis of PCR products flanking the sgRNA target derived from two control embryos or embryos injected with tfam sgRNA plus Cas9 protein. Sizes (left) of the ladder are indicated in base pairs (bp). (C) Representative sequences generated from PCR products amplified from individual control or tfam F0 mutant larvae that were TOPO cloned. PAM, protospacer adjacent motif (blue box); sgRNA has an estimated 100% mosaicism (based on 12 colonies from 4 different embryos). (D) Representative sequence chromatogram confirms a 9 bp deletion in tfam homozygous mutants: c.141_149del p.(Lys48_Pro50del) that was isolated by outcrossing F0 adults to WT (ZDR), isolation of F1 mutants, and incrossing of heterozygous adults


Figure S4. tfam homozygous mutant zebrafish adults show morphological differences compared to WT. (A) Representative images of adult zebrafish siblings at 4 months age. WT type males and females can be readily distinguished but tfam homozygous mutants cannot be characterized visually as male or female. (B) Quantification of the body length of WT and tfam homozygous mutant siblings measured at 4 months of age. n=7-9/condition; ****p<0.0001; unpaired t-test; error bars indicate standard deviation


Figure S5. tfam homozygous mutant zebrafish display a depletion in mitochondrial complex transcripts and mtDNA content. (A, B) Quantification of mtDNA copy number shows a significant depletion in tfam homozygous mutant adult head and tail tissues, two biological replicates; A, n=2 animals; B, n=3 animals. (C) Quantification of mt-nd1 and mt-co1 by qRT-PCR shows a significant reduction in tfam homozygous mutant adult gonad tissue. Repeated once with similar results, technical triplicates. (A-B-C). ****p < 0.0001; ***p<0.001, unpaired t-test; error bars indicate standard deviation


Figure S6. Validation of the morpholino used to ablate tfam in zebrafish. (A) Schematic shows the canonical zebrafish tfam transcript (GRCz11, ENSDART00000092009.6), green boxes, coding exons; untranslated regions (UTR), white boxes; black lines, introns. Arrow indicates morpholino (MO) target site at the exon 3 splice donor. (B) Agarose gel images show an aberrantly spliced transcript induced by tfam MO; cDNA was generated from total RNA isolated from 2 dpf embryos. The expected wild-type fragment is 378 bp. b-actin to was used to control for RNA integrity. (C) Representative sequences generated from individual colonies that were TOPO cloned from RT-PCR products generated from control and tfam morphants; exon 3 is excluded, resulting in mRNA lacking 71 bp in morphants. (D) Quantification of mtDNA copy number shows a significant depletion in tfam F0 mutants and morphants, two biological replicates; n=25 larvae per condition


Figure S7. In vivo complementation assay suggests that p.Arg232Cys impairs TFAM function. Zebrafish embryos were injected with tfam MO in the presence or absence of human TFAM mRNA; aged to 4 dpf; and pools of 25 larvae were harvested for quantification of mtDNA copy number using mt-nd1 as a mitochondrial marker and globin as a reference for nuclear genomic DNA. p.Pro178Leu is known to be pathogenic and has an absolute mtDNA content indistinguishable from MO alone. *p<0.05; **p<0.01; ns, not significant; data were generated from two biological replicates and compared with an unpaired t-test; error bars indicate standard deviation

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Ullah, F., Rauf, W., Khan, K. et al. A recessive variant in TFAM causes mtDNA depletion associated with primary ovarian insufficiency, seizures, intellectual disability and hearing loss. Hum Genet 140, 1733–1751 (2021).

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