Functional assessment of homozygous ALDH18A1 variants reveals alterations in amino acid and antioxidant metabolism.

Mono- and bi-allelic variants in ALDH18A1 cause a spectrum of human disorders associated with cutaneous and neurological findings that overlap with both cutis laxa and spastic paraplegia. ALDH18A1 encodes the bifunctional enzyme pyrroline-5-carboxylate synthetase (P5CS) that plays a role in the de novo biosynthesis of proline and ornithine. Here we characterize a previously unreported homozygous ALDH18A1 variant (p.Thr331Pro) in four affected probands from two unrelated families, and demonstrate broad-based alterations in amino acid and antioxidant metabolism. These four patients exhibit variable developmental delay, neurological deficits and loose skin. Functional characterization of the p.Thr331Pro variant demonstrated a lack of any impact on the steady-state level of the P5CS monomer or mitochondrial localization of the enzyme, but reduced incorporation of the monomer into P5CS oligomers. Using an unlabeled NMR-based metabolomics approach in patient fibroblasts and ALDH18A1-null human embryonic kidney cells expressing the variant P5CS, we identified reduced abundance of glutamate and several metabolites derived from glutamate, including proline and glutathione. Biosynthesis of the polyamine putrescine, derived from ornithine, was also decreased in patient fibroblasts, highlighting the functional consequence on another metabolic pathway involved in antioxidant responses in the cell. RNA sequencing of patient fibroblasts revealed transcript abundance changes in several metabolic and extracellular matrix-related genes, adding further insight into pathogenic processes associated with impaired P5CS function. Together these findings shed new light on amino acid and antioxidant pathways associated with ALDH18A1-related disorders, and underscore the value of metabolomic and transcriptomic profiling to discover new pathways that impact disease pathogenesis.

De Novo Variants in LMNB1 Cause Pronounced Syndromic Microcephaly and Disruption of Nuclear Envelope Integrity.

Lamin B1 plays an important role in the nuclear envelope stability, the regulation of gene expression, and neural development. Duplication of LMNB1, or missense mutations increasing LMNB1 expression, are associated with autosomal-dominant leukodystrophy. On the basis of its role in neurogenesis, it has been postulated that LMNB1 variants could cause microcephaly. Here, we confirm this hypothesis with the identification of de novo mutations in LMNB1 in seven individuals with pronounced primary microcephaly (ranging from -3.6 to -12 SD) associated with relative short stature and variable degree of intellectual disability and neurological features as the core symptoms. Simplified gyral pattern of the cortex and abnormal corpus callosum were noted on MRI of three individuals, and these individuals also presented with a more severe phenotype. Functional analysis of the three missense mutations showed impaired formation of the LMNB1 nuclear lamina. The two variants located within the head group of LMNB1 result in a decrease in the nuclear localization of the protein and an increase in misshapen nuclei. We further demonstrate that another mutation, located in the coil region, leads to increased frequency of condensed nuclei and lower steady-state levels of lamin B1 in proband lymphoblasts. Our findings collectively indicate that de novo mutations in LMNB1 result in a dominant and damaging effect on nuclear envelope formation that correlates with microcephaly in humans. This adds LMNB1 to the growing list of genes implicated in severe autosomal-dominant microcephaly and broadens the phenotypic spectrum of the laminopathies.

Deletion of the immunoglobulin domain of IL1RAPL1 results in nonsyndromic X-linked intellectual disability associated with behavioral problems and mild dysmorphism.

X-Linked intellectual disability accounts for a significant fraction of males with cognitive impairment. Many of these males present with a non-syndromic phenotype and presently mutations in 17 X-linked genes are associated with these patients. Mutations in IL1RAPL1 have been found in multiple families with non-syndromic X-linked intellectual disability. All of the published mutations predict loss of function of the protein. We have identified an additional two families with deletions of a portion of the gene that give rise to cognitive impairment, as well as some behavioral problems and mild dysmorphism. Our clinical findings better delineate the phenotypic spectrum associated with IL1RAPL1 mutations.

Severe multisystem pathology, metabolic acidosis, mitochondrial dysfunction, and early death associated with an X-linked AIFM1 variant.

Variants in the X-linked gene AIFM1 (apoptosis-inducing factor mitochondria-associated 1) are associated with a highly variable clinical presentation that encompasses motor neuropathy, ataxia, encephalopathies, deafness, and cognitive impairment. AIFM1 encodes a mitochondrial flavin adenine dinucleotide (FAD)-dependent nicotinamide adenine dinucleotide (NADH) oxidoreductase, with roles in the regulation of respiratory complex assembly and function, production of reactive oxygen species, and the coordination of a caspase-independent type of apoptosis known as parthanatos. In this report, we describe a missense AIFM1 variant (absent in reference population databases; c.506C > T, p.Pro169Leu) identified in the proband and sibling of a family with three affected males. The proband, his brother, and their maternal uncle all exhibited severe multisystem pathology, metabolic acidosis, and early demise. Metabolic testing on the proband revealed normal activity of the pyruvate dehydrogenase complex in skin fibroblasts. Absent or partial deficiency of cytochrome c oxidase was found in muscle fibers, however, supporting a Complex IV mitochondrial deficiency. Functional studies carried out on fibroblasts from the proband demonstrated reduced steady state levels of the AIFM1 protein, decreased Complex I subunit abundance, elevated sensitivity to the apoptosis inducer staurosporine, and increased nuclear condensation when grown in galactose-containing media. The reduced abundance of AIFM1 in the patient cells could not be stabilized with riboflavin or protease inhibitor treatment. Together, these findings suggest that the normal function of the AIFM1 gene product within mitochondria, and its response to apoptotic stimuli, are impaired by this variant, likely accounting for the severity of the phenotype seen in these patients. These findings also imply tissue-specific effects of this variant on different mitochondrial complexes. This study expands the genetic and phenotypic spectrum associated with AIFM1 variants, with the combination of exome sequencing and functional studies allowing a diagnosis to finally be confirmed for this family.

Functional analysis of a novel mutation in the TIMM8A gene that causes deafness-dystonia-optic neuronopathy syndrome.

BACKGROUND: The rare, X-linked neurodegenerative disorder, Mohr-Tranebjaerg syndrome (also called deafness-dystonia-optic neuronopathy [DDON] syndrome), is caused by mutations in the TIMM8A gene. DDON syndrome is characterized by dystonia, early-onset deafness, and various other neurological manifestations. The TIMM8A gene product localizes to the intermembrane space in mitochondria where it functions in the import of nuclear-encoded proteins into the mitochondrial inner membrane. Frameshifts or premature stops represent the majority of mutations in TIMM8A that cause DDON syndrome. However, missense mutations have also been reported that result in loss of the TIMM8A gene product. METHODS: We report a novel TIMM8A variant in a patient with DDON syndrome that alters the initiation codon and employed functional analyses to determine the significance of the variant and its impact on mitochondrial morphology. RESULTS: The novel base change in the TIMM8A gene (c.1A>T, p.Met1Leu) results in no detectable protein and a reduction in TIMM8A transcript abundance. We observed a commensurate decrease in the steady-state level of the Tim13 protein (the binding partner of Tim8a) but no decrease in TIMM13 transcripts. Patient fibroblasts exhibited elongation and/or increased fusion of mitochondria, consistent with prior reports. CONCLUSION: This case expands the spectrum of mutations that cause DDON syndrome and demonstrates effects on mitochondrial morphology that are consistent with prior reports.