Paul M. Fernhoff

Adaptor protein complex-4 (AP-4) deficiency causes a novel autosomal recessive cerebral palsy syndrome with microcephaly and intellectual disability

Background Cerebral palsy is a heterogeneous group of neurodevelopmental brain disorders resulting in motor and posture impairments often associated with cognitive, sensorial, and behavioural disturbances. Hypoxic–ischaemic injury, long considered the most frequent causative factor, accounts for fewer than 10% of cases, whereas a growing body of evidence suggests that diverse genetic abnormalities likely play a major role. Methods and results This report describes an autosomal recessive form of spastic tetraplegic cerebral palsy with profound intellectual disability, microcephaly, epilepsy and white matter loss in a consanguineous family resulting from a homozygous deletion involving AP4E1, one of the four subunits of the adaptor protein complex-4 (AP-4), identified by chromosomal microarray analysis. Conclusion These findings, along with previous reports of human and mouse mutations in other members of the complex, indicate that disruption of any one of the four subunits of AP-4 causes dysfunction of the entire complex, leading to a distinct ‘AP-4 deficiency syndrome’.

Population study of congenital hypothyroidism and associated birth defects, Atlanta, 1979-1992

Very little data are available from population-based studies on congenital hypothyroidism (CH) epidemiology and patterns of associated birth defects. By linking data from two population-based registries, we describe the epidemiology of CH and associated defects in Atlanta from 1979-1992. Cases included all infants with CH born from 1979-1992 to mothers residing in the metropolitan Atlanta area at the time of birth. We ascertained CH cases by reviewing newborn screening records and records of the Metropolitan Atlanta Congenital Defects Program (MACDP), a population-based registry of all serious birth defects diagnosed during a childs first year of life. We linked CH cases with MACDP records to ascertain the presence of serious birth defects among infants with CH. Of 97 infants identified with CH through newborn screening and/or MACDP (1:5,000 live births), 87 had primary CH and 10 had secondary. The rate of primary CH was higher among non-hispanic whites than among blacks (1:4,400 vs. 1:10,000) and among females compared with males (1:4,000 vs. 1:7,700). Among infants with primary CH, 77 had isolated CH, 3 had Down syndrome, and 7 had unrelated major structural defects. Based on Atlanta population rates of Down syndrome and major structural anomalies, we infer i) infants with Down syndrome have a 35-fold increased risk for primary CH compared with infants in the general population (P < .0001); ii) infants with primary CH have a 2.2-fold increased risk for major structural anomalies (P < .05). Because this is the first population study of CH in the United States in which data from two population-based registries were linked, the epidemiologic patterns and associated defects are more representative than those found in studies based on newborn screening records only.

Heparin cofactor II–thrombin complex: A biomarker of MPS disease

The mucopolysaccharidoses are a group of lysosomal storage disorders caused by defects in the degradation of glycosaminoglycans. Each disorder is characterized by progressive multi-system disease with considerable clinical heterogeneity. The clinical heterogeneity of these disorders is thought to be related to the degree of the metabolic block in glycosaminoglycan degradation which in turn is related to the underlying mutation at the respective locus. There are currently no objective means other than longitudinal clinical observation, or the detection of a recurrent genetic mutation to accurately predict the clinical course for an individual patient, particularly when diagnosed early. In addition, there are no specific disease biomarkers that reflect the total body burden of disease. The lack of specific biomarkers has made monitoring treatment responses and predicting disease course difficult in these disorders. The recent introduction of enzyme replacement therapy for MPS I, II, and VI highlights the need for objective measures of disease burden and disease responsiveness. We show that serum levels of heparin cofactor II-thrombin complex is a reliable biomarker of the mucopolysaccharidoses. Untreated patients have serum levels that range from 3- to 112-fold above control values. In a series of patients with varying severity of mucopolysaccharidosis I, the serum complex concentration was reflective of disease severity. In addition, serum heparin cofactor II-thrombin levels showed responsiveness to various treatment regimens. We propose that serum levels of heparin cofactor II-thrombin complex may provide an important assessment and monitoring tool for patients with mucopolysaccharidosis.

Defective urinary carnitine transport in heterozygotes for primary carnitine deficiency

Purpose: Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport and manifests as nonketotic hypoglycemia or skeletal or heart myopathy.Methods: To define the mechanisms producing partially reduced plasma carnitine levels in the parents of affected patients, we examined carnitine transport in vivo and in the fibroblasts of a new patient and his heterozygous parents.Results: Kinetic analysis of carnitine transport in fibroblasts revealed an absence of saturable carnitine transport in the probands cells and a partially impaired carnitine transport in fibroblasts from both parents, whose cells retained normal Km values toward carnitine (6–9 μM) but reduced Vmax. At steady state, normal fibroblasts accumulated carnitine to a concentration that was up to 80 times the extracellular value (0.5 μM). By contrast, cells from the proband had minimal carnitine accumulation, and cells from both parents had intermediate values of carnitine accumulation. Plasma carnitine levels were slightly below normal in both heterozygous, yet clinically normal, parents and in the paternal grandfather and the maternal grandmother. To define the mechanism producing partially decreased carnitine levels, we studied urinary carnitine losses in heterozygous parents compared with controls. Urinary losses increased linearly (P < 0.05) with plasma carnitine levels in normal controls. When urinary carnitine losses were normalized to plasma carnitine levels, a significant difference was observed between controls and heterozygous individuals (P < 0.01).Conclusions: These results indicate that fibroblasts from heterozygotes for primary carnitine deficiency have a decreased capacity to accumulate carnitine and that heterozygotes have increased urinary losses, which may contribute to their reduced plasma carnitine levels.

Medium chain acyl‐CoA dehydrogenase deficiency: Human genome epidemiology review

Medium chain acyl-CoA dehydrogenase (MCAD) is a tetrameric flavoprotein essential for the β-oxidation of medium chain fatty acids. MCAD deficiency (MCADD) is an inherited error of fatty acid metabolism. The gene for MCAD is located on chromosome one (1p31). One variant of the MCAD gene, G985A, a point mutation causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD protein, has been found in 90% of the alleles in MCADD patients identified retrospectively. There is a high frequency of MCADD among people of Northern European descent, which is believed to be due to a founder effect. MCADD is inherited in an autosomal recessive manner. Of patients clinically diagnosed with MCADD, 81% who have been identified retrospectively are homozygous for K304E, and 18% are compound heterozygotes for K304E. Clinical data on the probability of clinical disease indicates that MCADD patients are at risk for the following outcomes: hypoglycemia, vomiting, lethargy, encephalopathy, respiratory arrest, hepatomegaly, seizures, apnea, cardiac arrest, coma, and sudden and unexpected death. Long-term outcomes include developmental and behavioral disability, chronic muscle weakness, failure to thrive, cerebral palsy, and attention deficit disorder (ADD). Differences in clinical disease specific to allelic variants have not been documented. Factors that may increase risk for disease onset or modify disease severity are age when the first episode occurred, fasting, and presence of infection. Acute attacks must be treated immediately with appropriate intravenous doses of glucose. For those diagnosed, long-term management of the disease includes preventing stress caused by fasting and maintaining a high-carbohydrate, reduced-fat diet, and carnitine supplementation. Hospitalization costs attributable to morbidity and mortality from MCADD are unknown; MCADD is not a diagnosis in the International Classification of Disease, 10th Revision (ICD-10) codebook. Furthermore, the penetrance of the MCAD genotypes is unknown; there appears to be a substantial number of asymptomatic MCADD individuals and some uncertainty regarding which individuals will manifest symptoms and which individuals will remain asymptomatic. Several technologies are available to detect MCADD. Diagnostic technologies include DNA-based tests for K304E mutations using the polymerase chain reaction (PCR), and the detection of abnormal metabolites in urine. Screening technologies include tandem mass spectrometry (MS/MS), which detects abnormal metabolites mostly in blood. State programs are beginning to offer screening in newborns for MCADD using MS/MS. In addition, a private company currently offers voluntary supplemental newborn screening for MCADD to birthing centers.

Neonatal ethanol withdrawal: characteristics in clinically normal, nondysmorphic neonates.

Although neonatal withdrawal syndrome is often noted in infants of narcotics addicts, ethanol withdrawal has been reported only among neonates with fetal alcohol syndrome. To examine the possibility that ethanol withdrawal occurs more widely and to identify its characteristics, the behavior of eight neonates born to women who drank a mean of 21 ounces of absolute alcohol per week during gestation was compared with that of two contrast groups: 15 infants whose mothers drank an equivalent amount but stopped in the second trimester, and 29 infants whose mothers never drank. None of the 52 infants had fetal alcohol syndrome, and all were in good health. Neurobehavioral evaluation 3 days postnatally compared the groups for the occurrence of characteristic signs of withdrawal from central nervous system depressants. Whereas there was no difference in the frequency of withdrawal symptoms among infants of mothers who never drank (mean 1.4) or of mothers who stopped drinking (mean 1.8), infants of mothers who continued to drink (mean 4.7) had significantly more tremors, hypertonia, restlessness, excessive mouthing movements, unconsolable crying, and reflex abnormalities. By interfering with state control and interactive behaviors, withdrawal could affect mother-infant bonding as well as the conditions that foster cognitive and social development.

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.

Diagnosis of Fabry Disease via Analysis of Family History

Fabry disease is an X-linked lysosomal storage condition caused by a deficiency of α-galactosidase A. In order to determine the average number of family members who are diagnosed with Fabry disease following the diagnosis of a proband, four lysosomal storage disease centers across the United States reviewed the completed pedigrees of their Fabry disease patients. In addition, data from three Fabry disease families from other centers were submitted by patients directly. The pedigree review found 74 probands (54 males and 20 females) who had 357 diagnosed family members, of which 223 were female (60.5%) and 146 were male (39.5%). Analysis found that, on average, there were five family members diagnosed with Fabry disease for every proband. Now that enzyme replacement therapy (ERT) is available for the treatment of Fabry disease, this finding emphasizes the need for all health care professionals to ensure a detailed pedigree has been constructed for each patient affected by Fabry disease and to encourage testing and evaluation of all at-risk family members.

Acceptance of amniocentesis by low-income patients in an urban hospital☆☆☆

A study was made of increased accessibility of genetic services to low-income obstetric patients in Atlanta, Georgia. The proportion of black patients averaged 83%. Of 522 patients counseled from August, 1976, through 1978, 157 were offered amniocentesis, and 95 (61%) elected the procedure. For most of the patients (120, or 76%) who were eligible for amniocentesis, age (greater than or equal to 35 years at delivery) was an indication; and of these, only six (5%) had any prior knowledge of genetic risk. During the same time interval, 188 patients over 35 years of age who initiated prenatal care too late for prenatal diagnosis were counseled in the hospital after delivery; 101 (54%) indicated that they would have accepted amniocentesis. The conclusion was that (1) genetic services are acceptable to this socioeconomic group, and (2) accessibility and publicity are needed to promote utilization in this population.

Safety of extended treatment with sapropterin dihydrochloride in patients with phenylketonuria: Results of a phase 3b study

BACKGROUND Phenylketonuria (PKU) results from impaired breakdown of phenylalanine (Phe) due to deficient phenylalanine hydroxylase (PAH) activity. Sapropterin dihydrochloride (sapropterin, Kuvan®) is the only US- and EU-approved pharmaceutical version of naturally occurring 6R-BH(4), the cofactor required for PAH activity. Sapropterin enhances residual PAH activity in sapropterin-responsive PKU patients and, in conjunction with dietary management, helps reduce blood Phe concentrations for optimal control. Approval was based on the positive safety and efficacy results of four international clinical studies, the longest of which was 22 weeks in duration. OBJECTIVE To evaluate the safety of long-term treatment with sapropterin in PKU subjects who participated in previous Phase 3 sapropterin trials. METHODS PKU-008 was designed as a Phase 3b, multicenter, multinational, open-label, 3-year extension trial to evaluate the long-term safety of sapropterin in patients with PKU who were classified as sapropterin responders and participated in prior Phase 3 sapropterin studies: 111 subjects aged 4-50 years completed prior studies and were subsequently enrolled in study PKU-008. Routine safety monitoring was performed at 3-month intervals and included adverse event reporting, blood Phe monitoring, clinical laboratory evaluations, physical examinations and vital sign measurements. RESULTS Average exposure during PKU-008 was 658.7±221.3 days (range, 56-953; median, 595). The average total duration of participation in multiple studies (PKU-001, PKU-003, PKU-004, and PKU-008; or PKU-006 and PKU-008) was 799.0±237.5 days (range, 135-1151). The mean sapropterin dose was 16.2±4.7 mg/kg/day. Most adverse events were considered unrelated to treatment, were mild or moderate in severity, and were consistent with prior studies of sapropterin. No age-specific differences were observed in adverse event reporting. Three subjects discontinued treatment due to adverse events that were considered possibly or probably related to study treatment (one each of difficulty concentrating, decreased platelet count, and intermittent diarrhea). No deaths were reported. Of seven reported serious adverse events, one was considered possibly related to study treatment (gastroesophageal reflux). There were no laboratory or physical examination abnormalities requiring medical interventions. For most subjects, blood Phe concentrations were consistently within target range, confirming the durability of response in subjects undergoing extended treatment with sapropterin. CONCLUSION Sapropterin treatment was found to be safe and well tolerated at doses of 5 to 20mg/kg/day for an average exposure of 659 days. This study supports the safety and tolerability of sapropterin as long-term treatment for patients with PKU.