Dietrich Matern

Mutations in BCKD-kinase Lead to a Potentially Treatable Form of Autism with Epilepsy

A Fine Balance Intellectual and neurological disabilities can arise from diverse developmental aberrations. Novarino et al. (p. 394, published online 6 September; see the Perspective by Beaudet) have now determined the genetic basis for one such disorder for a small group of patients. Exome sequencing led to identification of mutations in a kinase BCKDK (Branched Chain Ketoacid Dehydrogenase Kinase) that regulates metabolism of branched-chain amino acids such as valine, leucine, and isoleucine. Mice with homozygous mutations in the BCKDK gene showed developmental and neurological abnormalities resembling those in certain mouse autism models. Analysis of transport mechanisms responsible for carrying amino acids across the blood-brain barrier revealed competition between the branched-chain amino acids and large neutral amino acids. Nutritional supplementation with extra branched-chain amino acids in the diet of mice carrying homozygous mutations in the BCKDK gene normalized their phenotype. When the balance of branched-chain amino acids transported into the brain goes awry, neurological deficits can ensue. Autism spectrum disorders are a genetically heterogeneous constellation of syndromes characterized by impairments in reciprocal social interaction. Available somatic treatments have limited efficacy. We have identified inactivating mutations in the gene BCKDK (Branched Chain Ketoacid Dehydrogenase Kinase) in consanguineous families with autism, epilepsy, and intellectual disability. The encoded protein is responsible for phosphorylation-mediated inactivation of the E1α subunit of branched-chain ketoacid dehydrogenase (BCKDH). Patients with homozygous BCKDK mutations display reductions in BCKDK messenger RNA and protein, E1α phosphorylation, and plasma branched-chain amino acids. Bckdk knockout mice show abnormal brain amino acid profiles and neurobehavioral deficits that respond to dietary supplementation. Thus, autism presenting with intellectual disability and epilepsy caused by BCKDK mutations represents a potentially treatable syndrome.

Lack of mitochondrial trifunctional protein in mice causes neonatal hypoglycemia and sudden death

Mitochondrial trifunctional protein (MTP) is a hetero-octamer of four alpha and four beta subunits that catalyzes the final three steps of mitochondrial long chain fatty acid beta-oxidation. Human MTP deficiency causes Reye-like syndrome, cardiomyopathy, or sudden unexpected death. We used gene targeting to generate an MTP alpha subunit null allele and to produce mice that lack MTP alpha and beta subunits. The Mtpa(-/-) fetuses accumulate long chain fatty acid metabolites and have low birth weight compared with the Mtpa(+/-) and Mtpa(+/+) littermates. Mtpa(-/-) mice suffer neonatal hypoglycemia and sudden death 6-36 hours after birth. Analysis of the histopathological changes in the Mtpa(-/-) pups revealed rapid development of hepatic steatosis after birth and, later, significant necrosis and acute degeneration of the cardiac and diaphragmatic myocytes. This mouse model documents that intact mitochondrial long chain fatty acid oxidation is essential for fetal development and for survival after birth. Deficiency of MTP causes fetal growth retardation, neonatal hypoglycemia, and sudden death.

Liver transplantation for glycogen storage disease types I, III, and IV

Abstract Glycogen storage disease (GSD) types I, III, and IV can be associated with severe liver disease. The possible development of hepatocellular carcinoma and/or hepatic failure make these GSDs potential candidates for liver transplantation. Early diagnosis and initiation of effective dietary therapy have dramatically improved the outcome of GSD type I by reducing the incidence of liver adenoma and renal insufficiency. Nine type I and 3 type III patients have received liver transplants because of poor metabolic control, multiple liver adenomas, or progressive liver failure. Metabolic abnormalities were corrected in all GSD type I and type III patients, while catch-up growth was reported only in two patients. Whether liver transplantation results in reversal and/or prevention of renal disease remains unclear. Neutropenia persisted in both GSDIb patients post liver transplantation necessitating continuous granulocyte colony stimulating factor treatment. Thirteen GSD type IV patients were liver transplanted because of progressive liver cirrhosis and failure. All but one patient have not had neuromuscular or cardiac complications during follow-up periods for as long as 13 years. Four have died within a week and 5 years after transplantation. Caution should be taken in selecting GSD type IV candidates for liver transplantation because of the variable phenotype, which may include life-limiting extrahepatic manifestations. It remains to be evaluated, whether a genotype-phenotype correlation exists for GSD type IV, which may aid in the decision making. Conclusion Liver transplantation should be considered for patients with glycogen storage disease who have developed liver malignancy or hepatic failure, and for type IV patients with the classical and progressive hepatic form.

Reduction of the false-positive rate in newborn screening by implementation of MS/MS-based second-tier tests: The Mayo Clinic experience (2004–2007)

SummaryThe continued expansion of newborn screening programmes to include additional conditions increases the responsibility of newborn screening laboratories to provide testing with the highest sensitivity and specificity to allow for identification of affected patients while minimizing the false-positive rate. Some assays and analytes are particularly problematic. Over recent years, our laboratory tried to improve this situation by developing second-tier tests to reduce false-positive results in the screening for congenital adrenal hyperplasia (CAH), tyrosinaemia type I, methylmalonic acidaemias, homocystinuria, and maple syrup urine disease (MSUD). Beginning in 2004, this approach was applied to Mayo’s newborn screening programme and resulted in a false-positive rate of 0.09%, a positive predictive value of 41%, and a positive detection rate of 1 affected case in 1672 babies screened.

A common mutation is associated with a mild, potentially asymptomatic phenotype in patients with isovaleric acidemia diagnosed by newborn screening

Isovaleric acidemia (IVA) is an inborn error of leucine metabolism that can cause significant morbidity and mortality. Since the implementation, in many states and countries, of newborn screening (NBS) by tandem mass spectrometry, IVA can now be diagnosed presymptomatically. Molecular genetic analysis of the IVD gene for 19 subjects whose condition was detected through NBS led to the identification of one recurring mutation, 932C-->T (A282V), in 47% of mutant alleles. Surprisingly, family studies identified six healthy older siblings with identical genotype and biochemical evidence of IVA. Our findings indicate the frequent occurrence of a novel mild and potentially asymptomatic phenotype of IVA. This has significant consequences for patient management and counseling.

Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots

BACKGROUND Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). METHODS We extracted 3/16-inch DBS punches with 300 microL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 microL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155-137) and 13C5-SUAC (m/z 160-142). Analysis time was 1.2 min. RESULTS SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13-81 micromol/L), with Tyr concentrations ranging from 65 to 293 micromol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 micromol/L. CONCLUSION The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

A Delphi clinical practice protocol for the management of very long chain acyl-CoA dehydrogenase deficiency

INTRODUCTION Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of oxidation of long chain fat, and can present as cardiomyopathy or fasting intolerance in the first months to years of life, or as myopathy in later childhood to adulthood. Expanded newborn screening has identified a relatively high incidence of this disorder (1:31,500), but there is a dearth of evidence-based outcomes data to guide the development of clinical practice protocols. This consensus protocol is intended to assist clinicians in the diagnosis and management of screen-positive newborns for VLCAD deficiency until evidence-based guidelines are available. METHOD The Oxford Centre for Evidence-based Medicine system was used to grade the literature review and create recommendations graded from A (evidence level of randomized clinical trials) to D (expert opinion). Delphi was used as the consensus tool. A panel of 14 experts (including clinicians, diagnostic laboratory directors and researchers) completed three rounds of survey questions and had a face-to-face meeting. RESULT Panelists reviewed the initial evaluation of the screen-positive infant, diagnostic testing and management of diagnosed patients. Grade C and D consensus recommendations were made in each of these three areas. The panel did not reach consensus on all issues, particularly in the dietary management of asymptomatic infants diagnosed by newborn screening.

Newborn screening for disorders of fatty-acid oxidation: experience and recommendations from an expert meeting.

Experience with new-born screening (NBS) for disorders of fatty-acid oxidation (FAOD) is now becoming available from an increasing number of programs worldwide. The spectrum of FAOD differs widely between ethnic groups. Incidence calculations from reports from Australia, Germany, and the USA of a total of 5,256,999 newborns give a combined incidence of all FAOD of approximately 1:9,300. However, it appears to be much lower in Asians. Consequently, a significant prevalence and evidence for a clear benefit of NBS is proven for medium-chain acyl-CoA dehydrogenase deficiency (MCAD) only in countries with a high percentage of Caucasians, with very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD) and long-chain 3-hydroxy acyl-CoA dehydrogenase deficiency (LCHAD) being additional candidates. The long-term benefit for many disorders has still to be evaluated and will require international collaboration, especially for the rarest disorders. Short-chain acyl-CoA dehydrogenase deficiency (SCAD) [as well as Systemic carnitine transporter deficiency (CTD) and dienoyl-CoA reductase deficiency (DE-RED)] are conditions of uncertain clinical significance, but most FAOD have a spectrum of clinical presentations (healthy–death). Confirmatory diagnostic procedures should be agreed upon to ensure international comparability of results and evidence-based modifications. The case of short-chain acyl-CoA dehydrogenase deficiency (SCAD) deficiency shows that even inclusion of conditions without a clearly known natural course may prove useful with respect to gain of knowledge and consecutive exclusion of a biochemical abnormality without clinical significance, although this line of argument implies the existence of structured follow-up programs and bears ethical controversies. As a final conclusion, the accumulated evidence suggests all FAOD should to be included into tandem mass spectrometry (MS/MS)-based NBS programs provided sufficient laboratory performance is guaranteed.

Acylcarnitine profile analysis

Disclaimer: These Technical Standards and Guidelines were developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patients record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular standard or guidelines was adopted, and to consider other relevant medical and scientific information that becomes available after that date.

Exome sequencing reveals a novel mutation for autosomal recessive non-syndromic mental retardation in the TECR gene on chromosome 19p13

Exome sequencing is a powerful tool for discovery of the Mendelian disease genes. Previously, we reported a novel locus for autosomal recessive non-syndromic mental retardation (NSMR) in a consanguineous family [Nolan, D.K., Chen, P., Das, S., Ober, C. and Waggoner, D. (2008) Fine mapping of a locus for nonsyndromic mental retardation on chromosome 19p13. Am. J. Med. Genet. A, 146A, 1414-1422]. Using linkage and homozygosity mapping, we previously localized the gene to chromosome 19p13. The parents of this sibship were recently included in an exome sequencing project. Using a series of filters, we narrowed the putative causal mutation to a single variant site that segregated with NSMR: the mutation was homozygous in five affected siblings but in none of eight unaffected siblings. This mutation causes a substitution of a leucine for a highly conserved proline at amino acid 182 in TECR (trans-2,3-enoyl-CoA reductase), a synaptic glycoprotein. Our results reveal the value of massively parallel sequencing for identification of novel disease genes that could not be found using traditional approaches and identifies only the seventh causal mutation for autosomal recessive NSMR.