Branched-chain α-ketoacid dehydrogenase deficiency (maple syrup urine disease): Treatment, biomarkers, and outcomes.

Over the past three decades, we studied 184 individuals with 174 different molecular variants of branched-chain α-ketoacid dehydrogenase activity, and here delineate essential clinical and biochemical aspects of the maple syrup urine disease (MSUD) phenotype. We collected data about treatment, survival, hospitalization, metabolic control, and liver transplantation from patients with classic (i.e., severe; n = 176), intermediate (n = 6) and intermittent (n = 2) forms of MSUD. A total of 13,589 amino acid profiles were used to analyze leucine tolerance, amino acid homeostasis, estimated cerebral amino acid uptake, quantitative responses to anabolic therapy, and metabolic control after liver transplantation. Standard instruments were used to measure neuropsychiatric outcomes. Despite advances in clinical care, classic MSUD remains a morbid and potentially fatal disorder. Stringent dietary therapy maintains metabolic variables within acceptable limits but is challenging to implement, fails to restore appropriate concentration relationships among circulating amino acids, and does not fully prevent cognitive and psychiatric disabilities. Liver transplantation eliminates the need for a prescription diet and safeguards patients from life-threatening metabolic crises, but is associated with predictable morbidities and does not reverse pre-existing neurological sequelae. There is a critical unmet need for safe and effective disease-modifying therapies for MSUD which can be implemented early in life. The biochemistry and physiology of MSUD and its response to liver transplantation afford key insights into the design of new therapies based on gene replacement or editing.

Recessive GM3 synthase deficiency: Natural history, biochemistry, and therapeutic frontier.

GM3 synthase, encoded by ST3GAL5, initiates synthesis of all downstream cerebral gangliosides. Here, we present biochemical, functional, and natural history data from 50 individuals homozygous for a pathogenic ST3GAL5 c.862C>T founder allele (median age 8.1, range 0.7-30.5 years). GM3 and its derivatives were undetectable in plasma. Weight and head circumference were normal at birth and mean Apgar scores were 7.7 ±â€¯2.0 (1 min) and 8.9 ±â€¯0.5 (5 min). Somatic growth failure, progressive microcephaly, global developmental delay, visual inattentiveness, and dyskinetic movements developed within a few months of life. Infantile-onset epileptic encephalopathy was characterized by a slow, disorganized, high-voltage background, poor state transitions, absent posterior rhythm, and spike trains from multiple independent cortical foci; >90% of electrographic seizures were clinically silent. Hearing loss affected cochlea and central auditory pathways and 76% of children tested failed the newborn hearing screen. Development stagnated early in life; only 13 (26%) patients sat independently (median age 30 months), three (6%) learned to crawl, and none achieved reciprocal communication. Incessant irritability, often accompanied by insomnia, began during infancy and contributed to high parental stress. Despite catastrophic neurological dysfunction, neuroimaging showed only subtle or no destructive changes into late childhood and hospitalizations were surprisingly rare (0.2 per patient per year). Median survival was 23.5 years. Our observations corroborate findings from transgenic mice which indicate that gangliosides might have a limited role in embryonic neurodevelopment but become vital for postnatal brain growth and function. These results have critical implications for the design and implementation of ganglioside restitution therapies.

Mass spectrometric quantification of plasma glycosphingolipids in human GM3 ganglioside deficiency.

BACKGROUND: Among Amish communities of North America, biallelic mutations of ST3GAL5 (c.694C > T) eliminate synthesis of GM3 and its derivative downstream a- and b-series gangliosides. Systemic ganglioside deficiency is associated with infantile onset psychomotor retardation, slow brain growth, intractable epilepsy, deafness, and cortical visual impairment. We developed a robust quantitative assay to simultaneously characterize glycan and ceramide moieties of plasma glycosphingolipids (GSLs) among ST3GAL5 c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control (n = 19) individuals. METHODS: Following extraction and saponification of total plasma lipids, GSLs were purified on a tC18 cartridge column, permethylated, and subjected to nanospray ionization mass spectrometry utilizing neutral loss scanning and data-dependent acquisition. Plasma GSLs were quantified against appropriate synthetic standards. RESULTS: Our method demonstrated linearity from 5 to 250 µl of plasma. Recovery of synthetic GSLs spiked into plasma was 99-104% with no matrix interference. Quantitative plasma GSL profiles discriminated among ST3GAL5 genotypes: GM3 and GD3 were undetectable in ST3GAL5 c.694C > T homozygotes, who had markedly elevated lactosylceramide (19.17 ±â€¯4.20 nmol/ml) relative to heterozygous siblings (9.62 ±â€¯2.46 nmol/ml) and wild type controls (6.55 ±â€¯2.16 nmol/ml). Children with systemic ganglioside deficiency had a distinctive shift in ceramide composition toward higher mass species. CONCLUSIONS: Our quantitative glycolipidomics method discriminates among ST3GAL5 c.694C > T genotypes, can reveal subtle structural heterogeneity, and represents a useful new strategy to diagnose and monitor GSL disorders in humans.

Genomic diagnostics within a medically underserved population: efficacy and implications.

PurposeWe integrated whole-exome sequencing (WES) and chromosomal microarray analysis (CMA) into a clinical workflow to serve an endogamous, uninsured, agrarian community.MethodsSeventy-nine probands (newborn to 49.8 years) who presented between 1998 and 2015 remained undiagnosed after biochemical and molecular investigations. We generated WES data for probands and family members and vetted variants through rephenotyping, segregation analyses, and population studies.ResultsThe most common presentation was neurological disease (64%). Seven (9%) probands were diagnosed by CMA. Family WES data were informative for 37 (51%) of the 72 remaining individuals, yielding a specific genetic diagnosis (n = 32) or revealing a novel molecular etiology (n = 5). For five (7%) additional subjects, negative WES decreased the likelihood of genetic disease. Compared to trio analysis, "family" WES (average seven exomes per proband) reduced filtered candidate variants from 22 ± 6 to 5 ± 3 per proband. Nineteen (51%) alleles were de novo and 17 (46%) inherited; the latter added to a population-based diagnostic panel. We found actionable secondary variants in 21 (4.2%) of 502 subjects, all of whom opted to be informed.ConclusionCMA and family-based WES streamline and economize diagnosis of rare genetic disorders, accelerate novel gene discovery, and create new opportunities for community-based screening and prevention in underserved populations.

Recessive nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum is caused by homozygous protein-truncating mutations of WDR73.

We describe a novel nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum among 30 children (ages 1.0 to 28 years) from diverse Amish demes. Children with nephrocerebellar syndrome had progressive microcephaly, visual impairment, stagnant psychomotor development, abnormal extrapyramidal movements and nephrosis. Fourteen died between ages 2.7 and 28 years, typically from renal failure. Post-mortem studies revealed (i) micrencephaly without polymicrogyria or heterotopia; (ii) atrophic cerebellar hemispheres with stunted folia, profound granule cell depletion, Bergmann gliosis, and signs of Purkinje cell deafferentation; (iii) selective striatal cholinergic interneuron loss; and (iv) optic atrophy with delamination of the lateral geniculate nuclei. Renal tissue showed focal and segmental glomerulosclerosis and extensive effacement and microvillus transformation of podocyte foot processes. Nephrocerebellar syndrome mapped to 700 kb on chromosome 15, which contained a single novel homozygous frameshift variant (WDR73 c.888delT; p.Phe296Leufs*26). WDR73 protein is expressed in human cerebral cortex, hippocampus, and cultured embryonic kidney cells. It is concentrated at mitotic microtubules and interacts with α-, ß-, and γ-tubulin, heat shock proteins 70 and 90 (HSP-70; HSP-90), and the carbamoyl phosphate synthetase 2/aspartate transcarbamylase/dihydroorotase multi-enzyme complex. Recombinant WDR73 p.Phe296Leufs*26 and p.Arg256Profs*18 proteins are truncated, unstable, and show increased interaction with α- and ß-tubulin and HSP-70/HSP-90. Fibroblasts from patients homozygous for WDR73 p.Phe296Leufs*26 proliferate poorly in primary culture and senesce early. Our data suggest that in humans, WDR73 interacts with mitotic microtubules to regulate cell cycle progression, proliferation and survival in brain and kidney. We extend the Galloway-Mowat syndrome spectrum with the first description of diencephalic and striatal neuropathology.

A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder.

We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G>A, p.Arg394His). KCNH7 c.1181G>A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G>A had the highest enrichment among individuals with bipolar spectrum disorder (χ(2) = 7.3) and the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031) and any major affective disorder (P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.