A systematic review on the biochemical threshold of mitochondrial genetic variants.

Mitochondrial DNA (mtDNA) variants cause a range of diseases from severe pediatric syndromes to aging-related conditions. The percentage of mtDNA copies carrying a pathogenic variant, variant allele frequency (VAF), must reach a threshold before a biochemical defect occurs, termed the biochemical threshold. Whether the often-cited biochemical threshold of >60% VAF is similar across mtDNA variants and cell types is unclear. In our systematic review, we sought to identify the biochemical threshold of mtDNA variants in relation to VAF by human tissue/cell type. We used controlled vocabulary terms to identify articles measuring oxidative phosphorylation (OXPHOS) complex activities in relation to VAF. We identified 76 eligible publications, describing 69, 12, 16, and 49 cases for complexes I, III, IV, and V, respectively. Few studies evaluated OXPHOS activities in diverse tissue types, likely reflective of clinical access. A number of cases with similar VAFs for the same pathogenic variant had varying degrees of residual activity of the affected complex, alluding to the presence of modifying variants. Tissues and cells with VAFs <60% associated with low complex activities were described, suggesting the possibility of a biochemical threshold of <60%. Using Kendall rank correlation tests, the VAF of the m.8993T > G variant correlated with complex V activity in skeletal muscle (τ = -0.58, P = 0.01, n = 13); however, no correlation was observed in fibroblasts (P = 0.7, n = 9). Our systematic review highlights the need to investigate the biochemical threshold over a wider range of VAFs in disease-relevant cell types to better define the biochemical threshold for specific mtDNA variants.

Further delineation of short-chain enoyl-CoA hydratase deficiency in the Pacific population.

Short-chain enoyl-coA hydratase (SCEH) deficiency due to biallelic pathogenic ECHS1 variants was first reported in 2014 in association with Leigh syndrome (LS) and increased S-(2-carboxypropyl)cysteine excretion. It is potentially treatable with a valine-restricted, high-energy diet and emergency regimen. Recently, Simon et al. described four Samoan children harbouring a hypomorphic allele (c.489G > A, p.Pro163=) associated with reduced levels of normally-spliced mRNA. This synonymous variant, missed on standard genomic testing, is prevalent in the Samoan population (allele frequency 0.17). Patients with LS and one ECHS1 variant were identified in NZ and Australian genomic and clinical databases. ECHS1 sequence data were interrogated for the c.489G > A variant and clinical data were reviewed. Thirteen patients from 10 families were identified; all had Pacific ancestry including Samoan, Maori, Cook Island Maori, and Tokelauan. All developed bilateral globus pallidi lesions, excluding one pre-symptomatic infant. Symptom onset was in early childhood, and was triggered by illness or starvation in 9/13. Four of 13 had exercise-induced dyskinesia, 9/13 optic atrophy and 6/13 nystagmus. Urine S-(2-carboxypropyl)cysteine-carnitine and other SCEH-related metabolites were normal or mildly increased. Functional studies demonstrated skipping of exon four and markedly reduced ECHS1 protein. These data provide further support for the pathogenicity of this ECHS1 variant which is also prevalent in Maori, Cook Island Maori, and Tongan populations (allele frequency 0.14-0.24). It highlights the need to search for a second variant in apparent heterozygotes with an appropriate phenotype, and has implications for genetic counselling in family members who are heterozygous for the more severe ECHS1 alleles. SYNOPSIS: Short-chain enoyl-CoA hydratase deficiency is a frequent cause of Leigh-like disease in Maori and wider-Pacific populations, due to the high carrier frequency of a hypomorphic ECHS1 variant c.489G > A, p.[Pro163=, Phe139Valfs*65] that may be overlooked by standard genomic testing.

Mitochondrial disease in New Zealand: a nationwide prevalence study.

BACKGROUND: The complexities of mitochondrial disease make epidemiological studies challenging, yet this information is important in understanding the healthcare burden and addressing service and educational needs. Existing studies are limited to quaternary centres or focus on a single genotype or phenotype and estimate disease prevalence at 12.5 per 100 000. New Zealand's (NZ) size and partially integrated national healthcare system make it amenable to a nationwide prevalence study. AIM: To estimate the prevalence of molecularly confirmed and suspected mitochondrial disease on 31 December 2015 in NZ. METHODS: Cases were identified from subspecialists and laboratory databases and through interrogation of the Ministry of Health National Minimum Dataset with a focus on presentations between 2000 and 2015. Patient records were reviewed, and those with a diagnosis of 'mitochondrial disease' who were alive and residing in NZ on the prevalence date were included. These were divided into molecularly confirmed and clinically suspected cases. Official NZ estimated resident population data were used to calculate prevalence. RESULTS: Seven hundred twenty-three unique national health index numbers were identified. Five hundred five were excluded. The minimum combined prevalence for mitochondrial disease was 4.7 per 100 000 (95% confidence interval (CI): 4.1-5.4). The minimum prevalence for molecularly confirmed and suspected disease was 2.9 (95% CI 2.4-3.4) and 1.8 (95% CI 1.4-2.2) cases per 100 000 respectively. CONCLUSIONS: Within the limitations of this study, comparison to similar prevalence studies performed by specialist referral centres suggests mitochondrial disease is underdiagnosed in NZ. This highlights a need for improved education and referral pathways for mitochondrial disease in NZ.

Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2.

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.

The management and clinical outcomes of pregnancies in women with urea cycle disorders: A review of the literature and results of an international survey.

An increasing number of women with urea cycle disorders (UCDs) are reaching child-bearing age and becoming pregnant. Improved diagnostics and increased awareness of inherited metabolic diseases has also led to more previously undetected women being diagnosed with a UCD during or shortly after pregnancy. Pregnancy increases the risk of acute metabolic decompensation with hyperammonemia-which can occur in any trimester, and/or the postpartum period, and may lead to encephalopathy, psychosis, coma, and even death, if not diagnosed promptly and treated appropriately. There are also (theoretical) concerns that a maternal UCD, or its treatment, may cause potential risks for the unborn child. Currently evidence on management and outcome of pregnancies in UCDs is limited to case reports and there are no clear guidelines. In order to inform management and investigate outcomes of pregnancies in women with a UCD, we performed a retrospective review of published cases and analyzed data collected from an international online survey. We conclude that, although risk during the intra- and postpartum period exists, multidisciplinary management by an experienced team and a prospective plan usually result in successful pregnancy, labor, delivery, and postpartum period. No deaths were reported in mothers managed accordingly. With the exception of male neonates with Ornithine Transcarbamylase deficiency, the clinical outcome of children born to mothers with UCDs appears positive, although follow-up is limited. The outcome for women presenting with a first acute metabolic decompensation during pregnancy or postpartum is less favorable. Deaths were associated with diagnostic delay/late management of hyperammonemia in previously undiagnosed women.

Metabolic disease in the Pacific: Lessons for indigenous populations.

Twenty-five percent of the New Zealand population is either Maori or Pacific and are thus indigenous to the region. The New Zealand National Metabolic Service has considerable experience in diagnosing and managing metabolic diseases in this population. The frequencies and phenotypes of inborn errors of metabolism in indigenous people differ from that in Western European populations. Metabolic services need to be aware of these local variations and adapt their screening and treatment protocols accordingly. Likewise, the services themselves need to adopt culturally appropriate practices. This includes an understanding of the language, ideally employment of indigenous people and targeting of the service to meet the needs of the people. Knowledge of the metabolic diseases common within particular ethnic groups is important for the rapid delivery of appropriate management. Newborn screening protocols need to reflect the local populations. With the advent of expanded newborn screening relatively benign forms of fatty acid oxidation disorders have been commonly encountered. This high prevalence may reflect a selective evolutionary advantage as similar conditions have been found in other ethnic groups with traditionally high fat and low carbohydrate diets. HLA haplotypes of indigenous populations are less represented in international stem cell transplant databanks thereby making the option of human stem cell transplant more challenging. The recent discovery that short-chain enoyl-CoA hydratase deficiency is particularly common in New Zealand with nearly a dozen cases identified this year suggests there is still a lot to learn regarding Maori and Pacific and indeed an indigenous metabolic disease.

New insights into carnitine-acylcarnitine translocase deficiency from 23 cases: Management challenges and potential therapeutic approaches.

Carnitine acyl-carnitine translocase deficiency (CACTD) is a rare autosomal recessive disorder of mitochondrial long-chain fatty-acid transport. Most patients present in the first 2 days of life, with hypoketotic hypoglycaemia, hyperammonaemia, cardiomyopathy or arrhythmia, hepatomegaly and elevated liver enzymes. Multi-centre international retrospective chart review of clinical presentation, biochemistry, treatment modalities including diet, subsequent complications, and mode of death of all patients. Twenty-three patients from nine tertiary metabolic units were identified. Seven attenuated patients of Pakistani heritage, six of these homozygous c.82G>T, had later onset manifestations and long-term survival without chronic hyperammonemia. Of the 16 classical cases, 15 had cardiac involvement at presentation comprising cardiac arrhythmias (9/15), cardiac arrest (7/15), and cardiac hypertrophy (9/15). Where recorded, ammonia levels were elevated in all but one severe case (13/14 measured) and 14/16 had hypoglycaemia. Nine classical patients survived longer-term-most with feeding difficulties and cognitive delay. Hyperammonaemia appears refractory to ammonia scavenger treatment and carglumic acid, but responds well to high glucose delivery during acute metabolic crises. High-energy intake seems necessary to prevent decompensation. Anaplerosis utilising therapeutic d,l-3-hydroxybutyrate, Triheptanoin and increased protein intake, appeared to improve chronic hyperammonemia and metabolic stability where trialled in individual cases. CACTD is a rare disorder of fatty acid oxidation with a preponderance to severe cardiac dysfunction. Long-term survival is possible in classical early-onset cases with long-chain fat restriction, judicious use of glucose infusions, and medium chain triglyceride supplementation. Adjunctive therapies supporting anaplerosis may improve longer-term outcomes.

The decision to discontinue screening for carnitine uptake disorder in New Zealand.

When screening for carnitine uptake disorder (CUD), the New Zealand (NZ) newborn screening (NBS) service identified infants as screen-positive if they had initial and repeat free carnitine (C0) levels of less than 5.0 µmol/L. Since 2006, the NBS service has identified two infants with biochemical and genetic features consistent with neonatal CUD and nine mothers with features consistent with maternal CUD. A review of the literature suggests that these nine women reflect less than half the true prevalence and that CUD is relatively common. However, the NZ results (two infants) suggest a very low sensitivity and positive predictive value of NBS. While patients presenting with significant disease due to CUD are well described, the majority of adults with CUD are asymptomatic. Nonetheless, treatment with high-dose oral L-carnitine is recommended. Compliance with oral L-carnitine is likely to be poor long term. This may represent a specific risk as treatment could repress the usual compensatory mechanisms seen in CUD, such that a sudden discontinuation of treatment may be dangerous. L-carnitine is metabolized to trimethylamine-N-oxide (TMAO) and treated patients have extremely high plasma TMAO levels. TMAO is an independent risk factor for atherosclerosis and, thus, caution should be exercised regarding long-term treatment with high-dose carnitine of asymptomatic patients who may have a biochemical profile without disease. Due to these concerns, the NZ Newborn Metabolic Screening Programme (NMSP) initiated a review via a series of advisory and governance committees and decided to discontinue screening for CUD.

The natural history of elevated tetradecenoyl-L-carnitine detected by newborn screening in New Zealand: implications for very long chain acyl-CoA dehydrogenase deficiency screening and treatment.

Very long chain acyl-CoA dehydrogenase deficiency (VLCADD, OMIM #201475) has been increasingly diagnosed since the advent of expanded newborn screening (NBS). Elevated levels of tetradecenoyl-L-carnitine (C14:1) in newborn screening blood spot samples are particularly common in New Zealand, however this has not translated into increased VLCADD clinical presentations. A high proportion of screen-positive cases in NZ are of Maori or Pacific ethnicity and positive for the c.1226C > T (p.Thr409Met) ACADVL gene variant. We performed a retrospective, blinded, case-control study of 255 cases, born between 2006 and 2013, with elevated NBS C14:1 levels between 0.9 and 2.4 µmol/L, below the NZ C14:1 notification cut-off of 2.5 µmol/L. Coded healthcare records were audited for cases and age- and ethnicity- matched controls. The clinical records of those with possible VLCADD-related symptoms were reviewed. The follow-up period was 6 months to 7 years. Two of 247 cases (0.8 %) had possible VLCADD-like symptoms while four of 247 controls (2 %) had VLCADD-like symptoms (p = 0.81). Maori were overrepresented (68 % of the cohort vs 15 % of population). Targeted analysis of the c.1226 locus revealed the local increase in screening C14:1 levels is associated with the c.1226C > T variant (97/152 alleles tested), found predominantly in Maori and Pacific people. There was no increase in clinically significant childhood disease, irrespective of ethnicity. The study suggests that children with elevated C14:1, between 0.9-2.4 µmol/L, on NBS are at very low risk of clinically significant childhood disease. A minimally interventional approach to managing these patients is indicated, at least in the New Zealand population.

Brain dopamine-serotonin vesicular transport disease presenting as a severe infantile hypotonic parkinsonian disorder.

Two male siblings from a consanguineous union presented in early infancy with marked truncal hypotonia, a general paucity of movement, extrapyramidal signs and cognitive delay. By mid-childhood they had made little developmental progress and remained severely hypotonic and bradykinetic. They developed epilepsy and had problems with autonomic dysfunction and oculogyric crises. They had a number of orthopaedic problems secondary to their hypotonia. Cerebrospinal fluid (CSF) neurotransmitters were initially normal, apart from mildly elevated 5-hydroxyindolacetic acid, and the children did not respond favourably to a trial of levodopa-carbidopa. The youngest died from respiratory complications at 10 years of age. Repeat CSF neurotransmitters in the older sibling at eight years of age showed slightly low homovanillic acid and 5-hydroxyindoleacetic acid levels. Whole-exome sequencing revealed a novel mutation homozygous in both children in the monoamine transporter gene SLC18A2 (p.Pro237His), resulting in brain dopamine-serotonin vesicular transport disease. This is the second family to be described with a mutation in this gene. Treatment with the dopamine agonist pramipexole in the surviving child resulted in mild improvements in alertness, communication, and eye movements. This case supports the identification of the causal mutation in the original case, expands the clinical phenotype of brain dopamine-serotonin vesicular transport disease and confirms that pramipexole treatment may lead to symptomatic improvement in affected individuals.

Mutations in SLC33A1 cause a lethal autosomal-recessive disorder with congenital cataracts, hearing loss, and low serum copper and ceruloplasmin.

Low copper and ceruloplasmin in serum are the diagnostic hallmarks for Menkes disease, Wilson disease, and aceruloplasminemia. We report on five patients from four unrelated families with these biochemical findings who presented with a lethal autosomal-recessive syndrome of congenital cataracts, hearing loss, and severe developmental delay. Cerebral MRI showed pronounced cerebellar hypoplasia and hypomyelination. Homozygosity mapping was performed and displayed a region of commonality among three families at chromosome 3q25. Deep sequencing and conventional sequencing disclosed homozygous or compound heterozygous mutations for all affected subjects in SLC33A1 encoding a highly conserved acetylCoA transporter (AT-1) required for acetylation of multiple gangliosides and glycoproteins. The mutations were found to cause reduced or absent AT-1 expression and abnormal intracellular localization of the protein. We also showed that AT-1 knockdown in HepG2 cells leads to reduced ceruloplasmin secretion, indicating that the low copper in serum is due to reduced ceruloplasmin levels and is not a sign of copper deficiency. The severity of the phenotype implies an essential role of AT-1 in proper posttranslational modification of numerous proteins, without which normal lens and brain development is interrupted. Furthermore, AT-1 defects are a new and important differential diagnosis in patients with low copper and ceruloplasmin in serum.

The risk of classical galactosaemia in newborns with borderline galactose metabolites on newborn screening.

Newborn screening (NBS) for classical galactosaemia (CG) facilitates early diagnosis and treatment to prevent life-threatening complications, but remains controversial, and screening protocols vary widely between programmes. False-negatives associated with first-tier screening of total galactose metabolites (TGAL) are infrequently reported; however, newborns with TGAL below the screening threshold have not been systematically studied. Following the diagnosis of CG in two siblings missed by NBS, a retrospective cohort study of infants with TGAL just below the cut-off (1.5 mmol/L blood) was conducted. Children born in New Zealand (NZ) from 2011 to 2019, with TGAL 1.0-1.49 mmol/L on NBS were identified from the national metabolic screening programme (NMSP) database, and clinical coding data and medical records were reviewed. GALT sequencing was performed if CG could not be excluded following review of medical records. 328 infants with TGAL 1.0-1.49 mmol/L on NBS were identified, of whom 35 had ICD-10 codes relevant to CG including vomiting, poor feeding, weight loss, failure to thrive, jaundice, hepatitis, Escherichia coli urinary tract infection, sepsis, intracranial hypertension and death. CG could be excluded in 34/35, due to documentation of clinical improvement with continued dietary galactose intake, or a clear alternative aetiology. GALT sequencing in the remaining individual confirmed Duarte-variant galactosaemia (DG). In conclusion, undiagnosed CG appears to be rare in those with TGAL 1.0-1.49 mmol/L on NBS; however, our recent experience with missed cases is nevertheless concerning. Further work is required to establish the optimum screening strategy, to maximize the early detection of CG without excess false-positives.

Novel patients with NHLRC2 variants expand the phenotypic spectrum of FINCA disease.

Purpose: FINCA disease (Fibrosis, Neurodegeneration and Cerebral Angiomatosis, OMIM 618278) is an infantile-onset neurodevelopmental and multiorgan disease. Since our initial report in 2018, additional patients have been described. FINCA is the first human disease caused by recessive variants in the highly conserved NHLRC2 gene. Our previous studies have shown that Nhlrc2-null mouse embryos die during gastrulation, indicating the essential role of the protein in embryonic development. Defect in NHLRC2 leads to cerebral neurodegeneration and severe pulmonary, hepatic and cardiac fibrosis. Despite having a structure suggestive of an enzymatic role and the clinical importance of NHLRC2 in multiple organs, the specific physiological role of the protein is unknown. Methods: The clinical histories of five novel FINCA patients diagnosed with whole exome sequencing were reviewed. Segregation analysis of the biallelic, potentially pathogenic NHLRC2 variants was performed using Sanger sequencing. Studies on neuropathology and NHLRC2 expression in different brain regions were performed on autopsy samples of three previously described deceased FINCA patients. Results: One patient was homozygous for the pathogenic variant c.442G > T, while the other four were compound heterozygous for this variant and two other pathogenic NHLRC2 gene variants. All five patients presented with multiorgan dysfunction with neurodevelopmental delay, recurrent infections and macrocytic anemia as key features. Interstitial lung disease was pronounced in infancy but often stabilized. Autopsy samples revealed widespread, albeit at a lower intensity than the control, NHLRC2 expression in the brain. Conclusion: This report expands on the characteristic clinical features of FINCA disease. Presentation is typically in infancy, and although patients can live to late adulthood, the key clinical and histopathological features are fibrosis, infection susceptibility/immunodeficiency/intellectual disability, neurodevelopmental disorder/neurodegeneration and chronic anemia/cerebral angiomatosis (hence the acronym FINCA) that enable an early diagnosis confirmed by genetic investigations.

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone.

Outcomes in mild hyperphenylalaninemia: a comparison with PKU and healthy controls across cognition, behaviour, and quality of life.

AIMS: Considering the cognitive, behavioural and quality of life (QoL) consequences of high phenylalanine levels in early treated phenylketonuria (PKU), this study examined whether monitoring and active management of individuals with the mild form of the condition hyperphenylalaninemia (HPA) would be advisable. METHOD: Six individuals (aged 6 to 15) with untreated HPA were compared with six age and gender matches with PKU, and six healthy controls on the Wechsler Intelligence Scale for Children, 5th edition; Wechsler Individual Achievement Test, 2nd edition; Trail-Making test; Contingency Naming Test; and Oral Fluency test. Self- and parent-report rating scales administered included the Conners Comprehensive Behavior Rating Scales; Behavior Rating Inventory of Executive Function, 2nd edition; the Pediatric Quality of Life Inventory, and the Phenylketonuria Quality of Life (PKU group only) questionnaires. RESULTS: Early treated PKU participants demonstrated normal intelligence, pointing to the efficacy of dietary management. Quality of life and behavioural difficulties were observed including more severe externalising problems. HPA participants showed normal ability, including executive ability. Power was limited by the small sample. CONCLUSION: This was the first study of the New Zealand population with HPA. While there was insufficient evidence to warrant treatment, there was also insufficient evidence to safely exclude the presence of cognitive impairment.

Genotype-phenotype correlations in CPT1A deficiency detected by newborn screening in Pacific populations.

Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a long chain fatty acid oxidation disorder, typically presenting with hypoketotic hypoglycaemia and liver dysfunction during fasting and intercurrent illness. Classical CPT1A deficiency is a rare disease, although a milder 'Arctic variant' (p.P479L) is common in the Inuit population. Since the introduction of expanded metabolic screening (EMS), the newborn screening programmes of Hawai'i and New Zealand (NZ) have detected a significant increase in the incidence of CPT1A deficiency. We report 22 individuals of Micronesian descent (12 in NZ and 10 in Hawai'i), homozygous for a CPT1A c.100T>C (p.S34P) variant detected by EMS or ascertained following diagnosis of a family member. No individuals with the Micronesian variant presented clinically with metabolic decompensation prior to diagnosis or during follow-up. Three asymptomatic homozygous adults were detected following the diagnosis of their children by EMS. CPT1A activity in cultured skin fibroblasts showed residual enzyme activity of 26% of normal controls. Secondly, we report three individuals from two unrelated Niuean families who presented clinically with symptoms of classic CPT1A deficiency, prior to the introduction of EMS. All were found to be homozygous for a CPT1A c.2122A>C (p.S708R) variant. CPT1A activity in fibroblasts of all three individuals was severely reduced at 4% of normal controls. Migration pressure, in part due to climate change may lead to increased frequency of presentation of Pacific peoples to regional metabolic services around the world. Knowledge of genotype-phenotype correlations in these populations will therefore inform counselling and treatment of those detected by newborn screening.

Megalencephalic leukoencephalopathy with cysts without MLC1 defect.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disease characterized by early infantile macrocephaly and delayed motor and cognitive deterioration. Magnetic resonance imaging (MRI) shows diffusely abnormal and swollen cerebral white matter and subcortical cysts. On follow-up, atrophy ensues. Approximately 80% of MLC patients have mutations in MLC1. We report 16 MLC patients without MLC1 mutations. Eight retained the classical clinical and MRI phenotype. The other 8 showed major MRI improvement. They lacked motor decline. Five had normal intelligence; 3 displayed cognitive deficiency. In conclusion, 2 phenotypes can be distinguished among the non-MLC1 mutated MLC patients: a classical and a benign phenotype.

Citrullinaemia type I: a common mutation in the Pacific Island population.

AIM: The aim of this study was to develop and apply a mutation screening protocol for the ASS1 gene in order to confirm the diagnosis of citrullinaemia type I in neonates with elevated citrulline on expanded newborn screening (E-NBS). METHODS: Three patients with an elevated citrulline level were identified via routine E-NBS between January and October 2008. Analysis of the ASS1 gene using a polymerase chain reaction and sequencing-based method was successfully applied to all three patients, together with a rapid mutation-specific detection method. Their clinical progress was followed for 16-22 months. RESULTS: All three patients were homozygous for a previously reported missense mutation, c.787G>A (p.Val263Met), associated with a mild or asymptomatic clinical course. CONCLUSIONS: As a consequence of E-NBS, an increasing number of neonates with elevated citrulline of uncertain clinical significance are being identified. Rapid sequence analysis of the ASS1 gene can be used to confirm citrullinaemia type I and, increasingly, to infer phenotypic severity. Homozygosity for the same mutation was found in all three patients despite non-consanguinity and variable Pacific Island origin. These data suggest that this mutation may be relatively prevalent in these ethnic groups and imply a possible founder effect.

Intellectual functioning in alpha-mannosidosis.

Alpha-mannosidosis is a rare inherited metabolic disorder (OMIM #248500) caused by mutations in the enzyme α-mannosidase encoded by the gene MAN2B1. Patients have distinct physical and developmental features, but only limited information regarding standardized cognitive functioning of patients has been published. Here we contribute intellectual ability scores (IQ) on 12 patients with alpha-mannosidosis (ages 8-59 years, 10 males, 2 females). In addition, a pooled analysis was performed with data collected from this investigation and 31 cases obtained from the literature, allowing a comprehensive analysis of intellectual functioning in this rare disease. The initial and pooled analyses show that patients with alpha-mannosidosis have variable degrees of intellectual disability but show decline in IQ with age, particularly during the first decade of life. Patients treated with hematopoietic stem cell transplantation tend to show stabilized cognitive abilities.

Untreated PKU Patients without Intellectual Disability: What Do They Teach Us?

Phenylketonuria (PKU) management is aimed at preventing neurocognitive and psychosocial dysfunction by keeping plasma phenylalanine concentrations within the recommended target range. It can be questioned, however, whether universal plasma phenylalanine target levels would result in optimal neurocognitive outcomes for all patients, as similar plasma phenylalanine concentrations do not seem to have the same consequences to the brain for each PKU individual. To better understand the inter-individual differences in brain vulnerability to high plasma phenylalanine concentrations, we aimed to identify untreated and/or late-diagnosed PKU patients with near-normal outcome, despite high plasma phenylalanine concentrations, who are still alive. In total, we identified 16 such cases. While intellectual functioning in these patients was relatively unaffected, they often did present other neurological, psychological, and behavioral problems. Thereby, these "unusual" PKU patients show that the classical symptomatology of untreated or late-treated PKU may have to be rewritten. Moreover, these cases show that a lack of intellectual dysfunction despite high plasma phenylalanine concentrations does not necessarily imply that these high phenylalanine concentrations have not been toxic to the brain. Also, these cases may suggest that different mechanisms are involved in PKU pathophysiology, of which the relative importance seems to differ between patients and possibly also with increasing age. Further research should aim to better distinguish PKU patients with respect to their cerebral effects to high plasma phenylalanine concentrations.