Renata C. Gallagher

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.

Evidence for the Role of PWCR1/HBII-85 C/D Box Small Nucleolar RNAs in Prader-Willi Syndrome

Prior work has suggested that loss of expression of one or more of the many C/D box small nucleolar RNAs (snoRNAs) encoded within the complex, paternally expressed SNRPN (small nuclear ribonuclear protein N) locus may result in the phenotype of Prader-Willi syndrome (PWS). We suggest that the minimal critical region for PWS is approximately 121 kb within the >460-kb SNRPN locus, bordered by a breakpoint cluster region identified in three individuals with PWS who have balanced reciprocal translocations and by the proximal deletion breakpoint of a familial deletion found in an unaffected mother, her three children with Angelman syndrome, and her father. The subset of SNRPN-encoded snoRNAs within this region comprises the PWCR1/HBII-85 cluster of snoRNAs and the single HBII-438A snoRNA. These are the only known genes within this region, which suggests that loss of their expression may be responsible for much or all of the phenotype of PWS. This hypothesis is challenged by findings in two individuals with PWS who have balanced translocations with breakpoints upstream of the proposed minimal critical region but whose cells were reported to express transcripts within it, adjacent to these snoRNAs. By use of real-time quantitative reverse-transcriptase polymerase chain reaction, we reassessed expression of these transcripts and of the snoRNAs themselves in fibroblasts of one of these patients. We find that the transcripts reported to be expressed in lymphoblast-somatic cell hybrids are not expressed in fibroblasts, and we suggest that the original results were misinterpreted. Most important, we show that the PWCR1/HBII-85 snoRNAs are not expressed in fibroblasts of this individual. These results are consistent with the hypothesis that loss of expression of the snoRNAs in the proposed minimal critical region confers much or all of the phenotype of PWS.

Clinical features and management issues in Mowat–Wilson syndrome†

Mowat–Wilson syndrome (MWS) is a relatively newly described multiple congenital anomaly/mental retardation syndrome. Haploinsufficiency of a gene termed ZFHX1B (also known as SIP1) on chromosome 2 is responsible for this condition, and clinical genetic testing for MWS recently became available. The majority of reports in the literature originate from Northern Europe and Australia. Here we report our clinical experience with 12 patients diagnosed with MWS within a 2‐year period of time in the United States, with particular emphasis on clinical characteristics and management strategies. Individuals with this condition have characteristic facial features, including microcephaly, hypertelorism, medially flared and broad eyebrows, prominent columella, pointed chin, and uplifted earlobes, which typically prompt the clinician to consider the diagnosis. Medical issues in our cohort of patients included seizures (75%) with no predeliction for any particular seizure type; agenesis of the corpus callosum (60% of our patients studied); congenital heart defects (75%), particularly involving the pulmonary arteries and/or valves; hypospadias (55% of males); severely impaired or absent speech (100% of individuals over 1 year of age) with relatively spared receptive language; and Hirschsprung disease (50%) or chronic constipation (25%). The incidence of MWS is unknown, but based on the number of patients identified in a short period of time within the US, it is likely greatly under recognized. MWS should be considered in any individual with severely impaired or absent speech, especially in the presence of seizures and anomalies involving the pulmonary arteries (particularly pulmonary artery sling) or pulmonary valves.

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.

AMMONIA CONTROL AND NEUROCOGNITIVE OUTCOME AMONG UREA CYCLE DISORDER PATIENTS TREATED WITH GLYCEROL PHENYLBUTYRATE

Glycerol phenylbutyrate is under development for treatment of urea cycle disorders (UCDs), rare inherited metabolic disorders manifested by hyperammonemia and neurological impairment. We report the results of a pivotal Phase 3, randomized, double‐blind, crossover trial comparing ammonia control, assessed as 24‐hour area under the curve (NH3‐AUC0‐24hr), and pharmacokinetics during treatment with glycerol phenylbutyrate versus sodium phenylbutyrate (NaPBA) in adult UCD patients and the combined results of four studies involving short‐ and long‐term glycerol phenylbutyrate treatment of UCD patients ages 6 and above. Glycerol phenylbutyrate was noninferior to NaPBA with respect to ammonia control in the pivotal study, with mean (standard deviation, SD) NH3‐AUC0‐24hr of 866 (661) versus 977 (865) μmol·h/L for glycerol phenylbutyrate and NaPBA, respectively. Among 65 adult and pediatric patients completing three similarly designed short‐term comparisons of glycerol phenylbutyrate versus NaPBA, NH3‐AUC0‐24hr was directionally lower on glycerol phenylbutyrate in each study, similar among all subgroups, and significantly lower (P < 0.05) in the pooled analysis, as was plasma glutamine. The 24‐hour ammonia profiles were consistent with the slow‐release behavior of glycerol phenylbutyrate and better overnight ammonia control. During 12 months of open‐label glycerol phenylbutyrate treatment, average ammonia was normal in adult and pediatric patients and executive function among pediatric patients, including behavioral regulation, goal setting, planning, and self‐monitoring, was significantly improved. Conclusion: Glycerol phenylbutyrate exhibits favorable pharmacokinetics and ammonia control relative to NaPBA in UCD patients, and long‐term glycerol phenylbutyrate treatment in pediatric UCD patients was associated with improved executive function (ClinicalTrials.gov NCT00551200, NCT00947544, NCT00992459, NCT00947297). (HEPATOLOGY 2012)

Establishing a consortium for the study of rare diseases: The Urea Cycle Disorders Consortium

The Urea Cycle Disorders Consortium (UCDC) was created as part of a larger network established by the National Institutes of Health to study rare diseases. This paper reviews the UCDCs accomplishments over the first 6years, including how the Consortium was developed and organized, clinical research studies initiated, and the importance of creating partnerships with patient advocacy groups, philanthropic foundations and biotech and pharmaceutical companies.

Succinyl-CoA ligase deficiency: a mitochondrial hepatoencephalomyopathy

This patient presented on the first day of life with pronounced lactic acidosis with an elevated lactate/pyruvate ratio. Urine organic acids showed Krebs cycle metabolites and mildly elevated methylmalonate and methylcitrate. The acylcarnitine profile showed elevated propionylcarnitine and succinylcarnitine. Amino acids showed elevated glutamic acid, glutamine, proline, and alanine. From the age 2 of mo on, she had elevated transaminases and intermittent episodes of liver failure. Liver biopsy showed steatosis and a decrease of mitochondrial DNA to 50% of control. She had bilateral sensorineural hearing loss. Over the course of the first 2 y of life, she developed a progressively severe myopathy with pronounced muscle weakness eventually leading to respiratory failure, Leigh disease, and recurrent hepatic failure. The hepatic symptoms and the metabolic parameters temporarily improved on treatment with aspartate, but neither muscle symptoms nor brain lesions improved. Laboratory testing revealed a deficiency of succinyl-CoA ligase enzyme activity and protein in fibroblasts because of a novel homozygous mutation in the SUCLG1 gene: c.40A>T (p.M14L). Functional analysis suggests that this methionine is more likely to function as the translation initiator methionine, explaining the pathogenic nature of the mutation. Succinyl-CoA ligase deficiency due to an SUCLG1 mutation is a new cause for mitochondrial hepatoencephalomyopathy.

Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin‐bound vitamin B12

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin‐bound cobalamin (holo‐TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo‐TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low‐density lipoprotein receptor type A‐like domain. Inserting the codon by site‐directed mutagenesis fully restored TCblR function. Hum Mutat 31:1–6, 2010.

High-frequency detection of deletions and variable rearrangements at the ornithine transcarbamylase (OTC) locus by oligonucleotide array CGH

Ornithine transcarbamylase (OTC) deficiency is an X-linked inborn error of metabolism characterized by impaired synthesis of citrulline from carbamylphosphate and ornithine. Previously reported data suggest that only approximately 80% of OTC deficiency (OTCD) patients have a mutation identified by OTC gene sequencing. To elucidate the molecular etiology in patients with clinical signs of OTCD and negative OTC sequencing, we subjected their DNA to array comparative genomic hybridization (aCGH) using a custom-designed targeted 44k oligonucleotide array. Whenever possible, parental DNA was analyzed to determine the inheritance or to rule out copy number variants in the OTC locus. DNA samples from a total of 70 OTCD patients were analyzed. Forty-three patients (43/70 or 61.5%) were found to have disease-causing point mutations in the OTC gene. The remaining 27 patients (27/70 or 38.5%) showed normal sequencing results or failure to amplify all or part of the OTC gene. Among those patients, eleven (11/70 or 15.7%) were found to have deletions ranging from 4.5kb to 10.6Mb, all involving the OTC gene. Sixteen OTCD patients (16/70 or 22.8%) had normal sequencing and oligoarray results. Analysis of the deletions did not reveal shared breakpoints, suggesting that non-homologous end joining or a replication-based mechanism might be responsible for the formation of the observed rearrangements. In summary, we demonstrate that approximately half of the patients with negative OTC sequencing may have OTC gene deletions readily identifiable by the targeted oligonucleotide-based aCGH. Thus, the test should be considered in OTC sequencing-negative patients with classic symptoms of the disease.

Functional Genetic Diversity in the High-Affinity Carnitine Transporter OCTN2 (SLC22A5)

Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n = 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, -207G>C. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (l-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p < 0.05). Further studies of the Phe17Leu polymorphism showed a reduced Vmax for l-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the -207G allele showed increased l-carnitine transport compared with the -207C/C homozygotes (p < 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.