Gunter Scharer

Folinic acid–responsive seizures are identical to pyridoxine-dependent epilepsy†

Folinic acid–responsive seizures and pyridoxine‐dependent epilepsy are two treatable causes of neonatal epileptic encephalopathy. The former is diagnosed by characteristic peaks on cerebrospinal fluid (CSF) monoamine metabolite analysis; its genetic basis has remained elusive. The latter is due to α‐aminoadipic semialdehyde (α‐AASA) dehydrogenase deficiency, associated with pathogenic mutations in the ALDH7A1 (antiquitin) gene. We report two patients whose CSF showed the marker of folinic acid–responsive seizures, but who responded clinically to pyridoxine. We performed genetic and biochemical testing of samples from these patients, and seven others, to determine the relation between these two disorders.

Genomics in Clinical Practice: Lessons from the Front Lines

This Commentary explores the challenges in launching a medical genomics clinic for whole genome sequencing and analysis of patient samples. The price of whole-genome and -exome sequencing has fallen to the point where these methods can be applied to clinical medicine. Here, we outline the lessons we have learned in converting a sequencing laboratory designed for research into a fully functional clinical program.

The genotypic and phenotypic spectrum of pyridoxine-dependent epilepsy due to mutations in ALDH7A1

Pyridoxine-dependent epilepsy is a disorder associated with severe seizures that may be caused by deficient activity of α-aminoadipic semialdehyde dehydrogenase, encoded by the ALDH7A1 gene, with accumulation of α-aminoadipic semialdehyde and piperideine-6-carboxylic acid. The latter reacts with pyridoxal-phosphate, explaining the effective treatment with pyridoxine. We report the clinical phenotype of three patients, their mutations and those of 12 additional patients identified in our clinical molecular laboratory. There were six missense, one nonsense, and five splice-site mutations, and two small deletions. Mutations c.1217_1218delAT, I431F, IVS-1(+2)T > G, IVS-2(+1)G > A, and IVS-12(+1)G > A are novel. Some disease alleles were recurring: E399Q (eight times), G477R (six times), R82X (two times), and c.1217_1218delAT (two times). A systematic review of mutations from the literature indicates that missense mutations cluster around exons 14, 15, and 16. Nine mutations represent 61% of alleles. Molecular modeling of missense mutations allows classification into three groups: those that affect NAD+ binding or catalysis, those that affect the substrate binding site, and those that affect multimerization. There are three clinical phenotypes: patients with complete seizure control with pyridoxine and normal developmental outcome (group 1) including our first patient; patients with complete seizure control with pyridoxine but with developmental delay (group 2), including our other two patients; and patients with persistent seizures despite pyridoxine treatment and with developmental delay (group 3). There is preliminary evidence for a genotype-phenotype correlation with patients from group 1 having mutations with residual activity. There is evidence from patients with similar genotypes for nongenetic factors contributing to the phenotypic spectrum.

An X-Linked Cobalamin Disorder Caused by Mutations in Transcriptional Coregulator HCFC1

Derivatives of vitamin B12 (cobalamin) are essential cofactors for enzymes required in intermediary metabolism. Defects in cobalamin metabolism lead to disorders characterized by the accumulation of methylmalonic acid and/or homocysteine in blood and urine. The most common inborn error of cobalamin metabolism, combined methylmalonic acidemia and hyperhomocysteinemia, cblC type, is caused by mutations in MMACHC. However, several individuals with presumed cblC based on cellular and biochemical analysis do not have mutations in MMACHC. We used exome sequencing to identify the genetic basis of an X-linked form of combined methylmalonic acidemia and hyperhomocysteinemia, designated cblX. A missense mutation in a global transcriptional coregulator, HCFC1, was identified in the index case. Additional male subjects were ascertained through two international diagnostic laboratories, and 13/17 had one of five distinct missense mutations affecting three highly conserved amino acids within the HCFC1 kelch domain. A common phenotype of severe neurological symptoms including intractable epilepsy and profound neurocognitive impairment, along with variable biochemical manifestations, was observed in all affected subjects compared to individuals with early-onset cblC. The severe reduction in MMACHC mRNA and protein within subject fibroblast lines suggested a role for HCFC1 in transcriptional regulation of MMACHC, which was further supported by the identification of consensus HCFC1 binding sites in MMACHC. Furthermore, siRNA-mediated knockdown of HCFC1 expression resulted in the coordinate downregulation of MMACHC mRNA. This X-linked disorder demonstrates a distinct disease mechanism by which transcriptional dysregulation leads to an inborn error of metabolism with a complex clinical phenotype.

Late-onset combined homocystinuria and methylmalonic aciduria (cblC) and neuropsychiatric disturbance

We report on the case of a 36‐year‐old Hispanic woman with a spinal cord infarct, who was subsequently diagnosed with methylmalonic aciduria and homocystinuria, cblC type (cblC). Mutation analysis revealed c.271dupA and c.482G > A mutations in the MMACHC gene. The patient had a past medical history significant for joint hypermobility, arthritis, bilateral cataracts, unilateral hearing loss, anemia, frequent urinary tract infections, and mental illness. There was no significant past history of mental retardation, failure to thrive, or seizure disorder as reported in classic cases of cblC. Prior to the thrombotic incident, the patient experienced increased paresthesia in the lower extremities, myelopathy, and impaired gait. Given her previous psychiatric history, she was misdiagnosed with malingering until hemiplegia and incontinence became apparent. The authors would like to emphasize the recognition of a neuropsychiatric presentation in late onset cblC. Ten other reported late onset cases with similar presentations are also reviewed.

Clinical impact of copy number variation analysis using high-resolution microarray technologies: advantages, limitations and concerns

Copy number variation (CNV) analysis has had a major impact on the field of medical genetics, providing a mechanism to identify disease-causing genomic alterations in an unprecedented number of diseases and phenotypes. CNV analysis is now routinely used in the clinical diagnostic laboratory, and has led to a significant increase in the detection of chromosomal abnormalities. These findings are used for prenatal decision making, clinical management and genetic counseling. Although a powerful tool to identify genomic alterations, CNV analysis may also result in the detection of genomic alterations that have unknown clinical significance or reveal unintended information. This highlights the importance of informed consent and genetic counseling for clinical CNV analysis. This review examines the advantages and limitations of CNV discovery in the clinical diagnostic laboratory, as well as the impact on the clinician and family.

TM4SF20 Ancestral Deletion and Susceptibility to a Pediatric Disorder of Early Language Delay and Cerebral White Matter Hyperintensities

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ~70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles.

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder

Background Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy. Methods and results We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNACys was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. Conclusions Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy.

The genetic basis of classic nonketotic hyperglycinemia due to mutations in GLDC and AMT

Purpose:The study’s purpose was to delineate the genetic mutations that cause classic nonketotic hyperglycinemia (NKH).Methods:Genetic results, parental phase, ethnic origin, and gender data were collected from subjects suspected to have classic NKH. Mutations were compared with those in the existing literature and to the population frequency from the Exome Aggregation Consortium (ExAC) database.Results:In 578 families, genetic analyses identified 410 unique mutations, including 246 novel mutations. 80% of subjects had mutations in GLDC. Missense mutations were noted in 52% of all GLDC alleles, most private. Missense mutations were 1.5 times as likely to be pathogenic in the carboxy terminal of GLDC than in the amino-terminal part. Intragenic copy-number variations (CNVs) in GLDC were noted in 140 subjects, with biallelic CNVs present in 39 subjects. The position and frequency of the breakpoint for CNVs correlated with intron size and presence of Alu elements. Missense mutations, most often recurring, were the most common type of disease-causing mutation in AMT. Sequencing and CNV analysis identified biallelic pathogenic mutations in 98% of subjects. Based on genotype, 15% of subjects had an attenuated phenotype. The frequency of NKH is estimated at 1:76,000.Conclusion:The 484 unique mutations now known in classic NKH provide a valuable overview for the development of genotype-based therapies.Genet Med 19 1, 104–111.

Neonatal liver failure: a genetic and metabolic perspective

Liver failure in newborns can present formidable diagnostic challenges. The presentation of neonatal liver failure is variable and the initial assessment is crucial in the determination of potentially treatable causes. We present a case of neonatal hemochromatosis, review genetic and metabolic causes of neonatal liver failure, and outline an updated differential diagnosis of neonatal liver failure. In addition, we propose a comprehensive initial work-up of neonatal liver failure, and review current treatments for neonatal hemochromatosis.