Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly / Novo mapeamento de mutações patogênicas do gene ASPM em famílias consanguíneas com microcefalia primária do Paquistão

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing", with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Characterization of ETFDH and PHGDH Mutations in a Patient with Mild Glutaric Aciduria Type II and Serine Deficiency.

Glutaric aciduria type II (GA-II) is a rare autosomal recessive disease caused by defects in electron transfer flavoprotein (ETF), ultimately causing insufficiencies in multiple acyl-CoA dehydrogenase (MAD). 3-phosphoglycerate dehydrogenase (3-PHGDH) deficiency, is another rare autosomal disorder that appears due to a defect in the synthesis of L-serine amino acid. Several mutations of ETFDH and PHGDH genes have been associated with different forms of GA-II and serine deficiency, respectively. In this study, we report a unique case of GA-II with serine deficiency using biochemical, genetic, and in silico approaches. The proband of Syrian descent had positive newborn screening (NBS) for GA-II. At two years of age, the patient presented with developmental regression, ataxia, and intractable seizures. Results of amino acid profiling demonstrated extremely low levels of serine. Confirmatory tests for GA-II and whole exome sequencing (WES) were performed to determine the etiology of intractable seizure. Sequencing results indicated a previously reported homozygous missense mutation, c.679 C>A (p.Pro227Thr) in the ETFDH gene and a novel missense homozygous mutation c.1219 T>C (p.Ser407Pro) in the PHGDH gene. In silico tools predicted these mutations as deleterious. Here, the clinical and biochemical investigations indicate that ETFDH:p.Pro227Thr and PHGDH:p.Ser407Pro variants likely underlie the pathogenesis of GA-II and serine deficiency, respectively. This study indicates that two rare autosomal recessive disorders should be considered in consanguineous families, more specifically in those with atypical presentation.

Plasma lipidome profiling of newborns with antenatal exposure to Zika virus.

The 2015-2016 Zika virus (ZIKV) outbreak in Brazil was remarkably linked to the incidence of microcephaly and other deleterious clinical manifestations, including eye abnormalities, in newborns. It is known that ZIKV targets the placenta, triggering an inflammatory profile that may cause placental insufficiency. Transplacental lipid transport is delicately regulated during pregnancy and deficiency on the delivery of lipids such as arachidonic and docosahexaenoic acids may lead to deficits in both brain and retina during fetal development. Here, plasma lipidome profiles of ZIKV exposed microcephalic and normocephalic newborns were compared to non-infected controls. Our results reveal major alterations in circulating lipids from both ZIKV exposed newborns with and without microcephaly relative to controls. In newborns with microcephaly, the plasma concentrations of hydroxyoctadecadienoic acid (HODE), primarily as 13-HODE isomer, derived from linoleic acid were higher as compared to normocephalic ZIKV exposed newborns and controls. Total HODE concentrations were also positively associated with levels of other oxidized lipids and several circulating free fatty acids in newborns, indicating a possible plasma lipidome signature of microcephaly. Moreover, higher concentrations of lysophosphatidylcholine in ZIKV exposed normocephalic newborns relative to controls suggest a potential disruption of polyunsaturated fatty acids transport across the blood-brain barrier of fetuses. The latter data is particularly important given the neurocognitive and neurodevelopmental abnormalities observed in follow-up studies involving children with antenatal ZIKV exposure, but normocephalic at birth. Taken together, our data reveal that plasma lipidome alterations associated with antenatal exposure to ZIKV could contribute to identification and monitoring of the wide spectrum of clinical phenotypes at birth and further, during childhood.

Microcephaly, Hypotonia, and Intracranial Calcifications in an 11-Week-Old Boy.

An 11-week-old unvaccinated, term Amish boy initially presented with poor feeding, microcephaly, failure to thrive, and developmental delays. His physical examination was significant for both weight and head circumference being less than the third percentile, and he was noted to have micrognathia, truncal hypotonia, and head lag. He was admitted to the pediatric hospital medicine service for further diagnostic evaluation. Laboratory studies assessing for endocrinological and metabolic etiologies yielded negative results, and imaging studies (including a chest radiograph, echocardiogram, and abdominal ultrasound) were normal. However, intracranial calcifications were noted on a head ultrasound. The etiology of his constellation of symptoms was initially thought to be infectious, but the ultimate diagnosis was not made until after discharge from the pediatric hospital medicine service.

Neonatal surveillance for congenital Zika infection during the 2016 microcephaly outbreak in Salvador, Brazil: Zika virus detection in asymptomatic newborns.

OBJECTIVE: To identify newborns with congenital Zika infection (CZI) at a maternity hospital in Salvador, Brazil, during the 2016 microcephaly outbreak. METHODS: A prospective study enrolled microcephalic and normocephalic newborns with suspected CZI between January and December 2016. Serology (immunoglobulins IgM and IgG) and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) for the Zika virus were performed. Demographic and clinical characteristics of newborns with and without microcephaly were compared. RESULTS: Of the 151 newborns enrolled, 32 (21.2%) were classified as microcephalic. The majority of these cases were born between January and May 2016. IgM and IgG Zika virus antibodies were detected in 5 (23.8%) and 17 (80.9%) microcephalic newborn blood samples, respectively. Six (24%) microcephalic newborns tested positive for Zika virus by RT-qPCR in urine or placenta samples. Thirteen (11.8%) normocephalic newborns also tested positive for Zika virus by PCR in urine, plasma, or placenta samples, while IgM antibodies against Zika were detected in 4 (4.2%) others. CONCLUSIONS: Identification of 17 normocephalic CZI cases, confirmed by IgM serology or RT-qPCR for Zika virus, provides evidence that CZI can present asymptomatically at birth. This finding highlights the need for prenatal and neonatal screening for Zika virus in endemic regions.

Emergence of the Asian lineage of Zika virus in Angola: an outbreak investigation.

BACKGROUND: Zika virus infections and suspected microcephaly cases have been reported in Angola since late 2016, but no data are available about the origins, epidemiology, and diversity of the virus. We aimed to investigate the emergence and circulation of Zika virus in Angola. METHODS: Diagnostic samples collected by the Angolan Ministry of Health as part of routine arboviral surveillance were tested by real-time reverse transcription PCR by the Instituto Nacional de Investigação em Saúde (Ministry of Health, Luanda, Angola). To identify further samples positive for Zika virus and appropriate for genomic sequencing, we also tested samples from a 2017 study of people with HIV in Luanda. Portable sequencing was used to generate Angolan Zika virus genome sequences from three people positive for Zika virus infection by real-time reverse transcription PCR, including one neonate with microcephaly. Genetic and mobility data were analysed to investigate the date of introduction and geographical origin of Zika virus in Angola. Brain CT and MRI, and serological assays were done on a child with microcephaly to confirm microcephaly and assess previous Zika virus infection. FINDINGS: Serum samples from 54 people with suspected acute Zika virus infection, 76 infants with suspected microcephaly, 24 mothers of infants with suspected microcephaly, 336 patients with suspected dengue virus or chikungunya virus infection, and 349 samples from the HIV study were tested by real-time reverse transcription PCR. Four cases identified between December, 2016, and June, 2017, tested positive for Zika virus. Analyses of viral genomic and human mobility data suggest that Zika virus was probably introduced to Angola from Brazil between July, 2015, and June, 2016. This introduction probably initiated local circulation of Zika virus in Angola that continued until at least June, 2017. The infant with microcephaly in whom CT and MRI were done had brain abnormalities consistent with congenital Zika syndrome and serological evidence for Zika virus infection. INTERPRETATION: Our analyses show that autochthonous transmission of the Asian lineage of Zika virus has taken place in Africa. Zika virus surveillance and surveillance of associated cases of microcephaly throughout the continent is crucial. FUNDING: Royal Society, Wellcome Trust, Global Challenges Research Fund (UK Research and Innovation), Africa Oxford, John Fell Fund, Oxford Martin School, European Research Council, Departamento de Ciência e Tecnologia/Ministério da Saúde/National Council for Scientific and Technological Development, and Ministério da Educação/Coordenação de Aperfeicoamento de Pessoal de Nível Superior.

Disturbed phospholipid metabolism in serine biosynthesis defects revealed by metabolomic profiling.

Serine biosynthesis defects are autosomal recessive metabolic disorders resulting from the deficiency of any of the three enzymes involved in de novo serine biosynthesis, specifically phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). In this study, we performed metabolomic profiling on 4 children with serine biosynthesis defects; 3 with PGDH deficiency and 1 with PSAT deficiency. The evaluations were performed at baseline and with serine and glycine supplementation. Metabolomic profiling performed at baseline showed low phospholipid species, including glycerophosphocholine, glycerophosphoethanolamine, and sphingomyelin. All children had low serine and glycine as expected. Low glycerophosphocholine compounds were found in 4 children, low glycerophosphoethanolamine compounds in 3 children, and low sphingomyelin species in 2 children. Metabolic profiling with serine and glycine supplementation showed normalization of most of the low phospholipid compounds in the 4 children. Phospholipids are the major component of plasma and intracellular membranes, and phosphatidylcholine is the most abundant phospholipid of all mammalian cell types and subcellular organelles. Phosphatidylcholine is of particular importance for the nervous system, where it is essential for neuronal differentiation. The observed low phosphatidylcholine species in children with serine biosynthesis defects that improved after serine supplementation, supports the role of serine as a significant precursor for phosphatidylcholine. The vital role that phosphatidylcholine has during neuronal differentiation and the pronounced neurological manifestations in serine biosynthesis defects suggest that phosphatidylcholine deficiency occurring secondary to serine deficiency may have a significant contribution to the development of the neurological manifestations in individuals with serine biosynthesis defects.

Systemic inflammation on postnatal days 21 and 28 and indicators of brain dysfunction 2years later among children born before the 28th week of gestation.

BACKGROUND: Systemic inflammation during the first two postnatal weeks in extremely preterm newborns (<28weeks gestation) has been associated with an increased risk of neurodevelopmental dysfunctions. Little is known, however, about the relationship between systemic inflammation during the third and fourth postnatal weeks and subsequent development. METHODS: We measured the concentrations of 16 inflammation-related proteins in blood spots collected on postnatal days 21 (N=749) and 28 (N=697) from infants born before the 28th week of gestation and assessed at age 2years. We then sought the developmental correlates of top quartile concentrations for gestational age and day the specimen was collected. Odds ratios and 95% confidence intervals were calculated from regular or multinomial logistic regression models (as appropriate). RESULTS: Top quartile concentrations of CRP, IL-1ß, IL-6, IL-6R, TNF-R2, IL-8, ICAM-1, and TSH on both days 21 and 28 were associated with ventriculomegaly (when in the NICU) and microcephaly at age 2years. Top quartile concentrations of CRP, SAA, IL-6, TNF-R2, IL-8, and ICAM-1 were associated with mental development index (MDI) of the Bayley-II<55, while top quartile concentrations of CRP, TNF-α (inversely), IL-8, and ICAM-1 were associated with psychomotor development index (PDI)<55 CONCLUSION: Extremely preterm newborns who had systemic inflammation during the third and fourth postnatal weeks were at increased risk of ventriculomegaly during the months after birth, and of microcephaly, and low Bayley Scale scores at 2years old.

Clinical and genetic features of dyskeratosis congenita, cryptic dyskeratosis congenita, and Hoyeraal-Hreidarsson syndrome in Japan.

Dyskeratosis congenita (DKC) is an inherited bone marrow failure (BMF) syndrome typified by reticulated skin pigmentation, nail dystrophy, and mucosal leukoplakia. Hoyeraal-Hreidarsson syndrome (HHS) is considered to be a severe form of DKC. Unconventional forms of DKC, which develop slowly in adulthood but without the physical anomalies characteristic of DKC (cryptic DKC), have been reported. Clinical and genetic features of DKC have been investigated in Caucasian, Black, and Hispanic populations, but not in Asian populations. The present study aimed to determine the clinical and genetic features of DKC, HHS, and cryptic DKC among Japanese patients. We analyzed 16 patients diagnosed with DKC, three patients with HHS, and 15 patients with cryptic DKC. We found that platelet count was significantly more depressed than neutrophil count or hemoglobin value in DKC patients, and identified DKC patients with large deletions in the telomerase reverse transcriptase and cryptic DKC patients with RTEL1 mutations on both alleles. This led to some patients previously considered to have unclassifiable BMF being diagnosed with cDKC through identification of new gene mutations. It thus seems important from a clinical viewpoint to re-examine the clinical characteristics, frequency of genetic mutations, and treatment efficacy in DKC, HHS, and cDKC.

A partially inactivating mutation in the sodium-dependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome.

The major pathway by which the brain obtains essential omega-3 fatty acids from the circulation is through a sodium-dependent lysophosphatidylcholine (LPC) transporter (MFSD2A), expressed in the endothelium of the blood-brain barrier. Here we show that a homozygous mutation affecting a highly conserved MFSD2A residue (p.Ser339Leu) is associated with a progressive microcephaly syndrome characterized by intellectual disability, spasticity and absent speech. We show that the p.Ser339Leu alteration does not affect protein or cell surface expression but rather significantly reduces, although not completely abolishes, transporter activity. Notably, affected individuals displayed significantly increased plasma concentrations of LPCs containing mono- and polyunsaturated fatty acyl chains, indicative of reduced brain uptake, confirming the specificity of MFSD2A for LPCs having mono- and polyunsaturated fatty acyl chains. Together, these findings indicate an essential role for LPCs in human brain development and function and provide the first description of disease associated with aberrant brain LPC transport in humans.

Elevated endogenous erythropoietin concentrations are associated with increased risk of brain damage in extremely preterm neonates.

BACKGROUND: We sought to determine, in very preterm infants, whether elevated perinatal erythropoietin (EPO) concentrations are associated with increased risks of indicators of brain damage, and whether this risk differs by the co-occurrence or absence of intermittent or sustained systemic inflammation (ISSI). METHODS: Protein concentrations were measured in blood collected from 786 infants born before the 28th week of gestation. EPO was measured on postnatal day 14, and 25 inflammation-related proteins were measured weekly during the first 2 postnatal weeks. We defined ISSI as a concentration in the top quartile of each of 25 inflammation-related proteins on two separate days a week apart. Hypererythropoietinemia (hyperEPO) was defined as the highest quartile for gestational age on postnatal day 14. Using logistic regression and multinomial logistic regression models, we compared risks of brain damage among neonates with hyperEPO only, ISSI only, and hyperEPO+ISSI, to those who had neither hyperEPO nor ISSI, adjusting for gestational age. RESULTS: Newborns with hyperEPO, regardless of ISSI, were more than twice as likely as those without to have very low (< 55) Mental (OR 2.3; 95% CI 1.5-3.5) and/or Psychomotor (OR 2.4; 95% CI 1.6-3.7) Development Indices (MDI, PDI), and microcephaly at age two years (OR 2.4; 95%CI 1.5-3.8). Newborns with both hyperEPO and ISSI had significantly increased risks of ventriculomegaly, hemiparetic cerebral palsy, microcephaly, and MDI and PDI < 55 (ORs ranged from 2.2-6.3), but not hypoechoic lesions or other forms of cerebral palsy, relative to newborns with neither hyperEPO nor ISSI. CONCLUSION: hyperEPO, regardless of ISSI, is associated with elevated risks of very low MDI and PDI, and microcephaly, but not with any form of cerebral palsy. Children with both hyperEPO and ISSI are at higher risk than others of very low MDI and PDI, ventriculomegaly, hemiparetic cerebral palsy, and microcephaly.

The breadth and type of systemic inflammation and the risk of adverse neurological outcomes in extremely low gestation newborns.

BACKGROUND: We hypothesized that the risk of brain damage in extremely preterm neonates increases with the breadth and type of systemic inflammation, indexed by the number of elevated inflammation-related proteins and the number of functional categories of inflammation-related proteins exhibiting an elevated concentration. METHODS: In blood from 881 infants born before 28 weeks gestation, we measured the concentrations of 25 inflammation-related proteins, representing six functional categories (cytokines, chemokines, growth factors, adhesion molecules, metalloproteinases, and liver-produced acute phase reactant proteins) on postnatal days 1, 7, and 14. We evaluated associations between the number and type of proteins whose concentrations were elevated on two separate occasions a week apart and the diagnoses of ventriculomegaly as a neonate, and at 2 years, microcephaly, impaired early cognitive functioning, cerebral palsy, and autism risk as assessed with the Modified Checklist for Autism in Toddlers screen, and in a subset of these children from 12 of 14 sites (n = 826), an attention problem identified with the Child Behavior Checklist. RESULTS: The risk of abnormal brain structure and function overall was increased among children who had recurrent and/or persistent elevations of the 25 proteins. The risk for most outcomes did not rise until at least four proteins in at least two functional categories were elevated. When we focused our analysis on 10 proteins previously found to be associated consistently with neurological outcomes, we found the risk of low Mental Development Index on the Bayley Scales of Infant Development-II, microcephaly, and a Child Behavior Checklist-defined attention problem increased with higher numbers of these recurrently and/or persistently elevated proteins. INTERPRETATION: Increasing breadth of early neonatal inflammation, indexed by the number of protein elevations or the number of protein functional classes elevated, is associated with increasing risk of disorders of brain structure and function among infants born extremely preterm.

Are preterm newborns who have relative hyperthyrotropinemia at increased risk of brain damage?

BACKGROUND: We sought to disentangle the contributions of hyperthyrotropinemia (an indicator of thyroid dysfunction) (HTT) and intermittent or sustained systemic inflammation (ISSI) to structural and functional indicators of brain damage. METHODS: We measured the concentrations of thyroid-stimulating hormone (TSH) on day 14 and of 25 inflammation-related proteins in blood collected during the first 2 postnatal weeks from 786 infants born before the 28th week of gestation who were not considered to have hypothyroidism. We defined hyperthyrotropinemia (HTT) as a TSH concentration in the highest quartile for gestational age on postnatal day 14 and ISSI was defined as a concentration in the top quartile for gestational age of a specific inflammation-related protein on 2 separate days a week apart during the first 2 postnatal weeks. We first assessed the risk of brain damage indicators by comparing 1) neonates who had HTT to those without (regardless of ISSI) and 2) neonates with HTT only, ISSI only, or HTT+ISSI to those who were exposed to neither HTT nor ISSI. RESULTS: In univariable models that compared those with HTT to those without, HTT was not significantly associated with any indicator of brain damage. In models that compared HTT only, ISSI only, and HTT+ISSI to those with neither, children with ISSI only or with HTT+ISSI were at significantly higher risk of ventriculomegaly [odds ratios (ORs) 2-6], whereas those with HTT only were at significantly reduced risk of a hypoechoic lesion (ORs 0.2-0.4). Children with HTT only had a higher risk of quadriparesis and those with ISSI alone had a higher risk of hemiparesis (ORs 1.6-2.4). Elevated risk of a very low mental development score was associated with both ISSI only and HTT+ISSI, whereas a very low motor development score and microcephaly were associated with HTT+ISSI. CONCLUSIONS: The association of HTT with increased or decreased risk of indicators of brain damage depends on the presence or absence of ISSI.

An update on serine deficiency disorders.

Serine deficiency disorders are caused by a defect in one of the three synthesising enzymes of the L-serine biosynthesis pathway. Serine deficiency disorders give rise to a neurological phenotype with psychomotor retardation, microcephaly and seizures in newborns and children or progressive polyneuropathy in adult patients. There are three defects that cause serine deficiency of which 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, the defect affecting the first step in the pathway, has been reported most frequently. The other two disorders in L-serine biosynthesis phosphoserine aminotransferase (PSAT) deficiency and phosphoserine phosphatase (PSP) deficiency have been reported only in a limited number of patients. The biochemical hallmarks of all three disorders are low concentrations of serine in cerebrospinal fluid and plasma. Prompt recognition of affected patients is important, since serine deficiency disorders are treatable causes of neurometabolic disorders. The use of age-related reference values for serine in CSF and plasma can be of great help in establishing a correct diagnosis of serine deficiency, in particular in newborns and young children.

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients.

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

Early postnatal blood concentrations of inflammation-related proteins and microcephaly two years later in infants born before the 28th post-menstrual week.

BACKGROUND: To evaluate if concentrations of inflammation-related proteins were elevated in early postnatal blood specimens of preterm newborns who two years later had a small head. METHODS: We measured 25 proteins in blood collected on days 1, 7, and 14 from 839 infants born before the 28th week of gestation and whose head circumference was measured at birth and near 24-months post-term equivalent. We excluded children whose birth head circumference was at or below the third centile. A protein concentration was considered elevated if it was in the highest quartile for gestational age and the day the specimen was obtained. FINDINGS: When proteins were evaluated individually, elevated concentrations of SAA on day 1 and five proteins on day 14, IL-6, TNF-R2, IL-8, MCP-1, and ICAM-1 were associated with significantly increased risk of microcephaly (head circumference Z-score <-2). The ten proteins whose elevated concentrations on two separate days a week apart predicted microcephaly, but did not do so when elevated on only one of these days were CRP, SAA, IL-1ß, IL-6, TNF-α, IL-8, MCP-1, ICAM-1, E-SEL, IGFBP-1. Elevated protein concentrations weakly predicted a less severe reduction in head circumference (Z-score ≥ -2, < -1). INTERPRETATION: Concentrations of inflammation-related proteins in the circulation shortly after preterm birth provide information about the risk of a reduced head circumference more than two years later. FUNDING: The ELGAN Study was supported by a cooperative agreement with the National Institute of Neurological Disorders and Stroke of the United States.

Amish microcephaly: Long-term survival and biochemical characterization.

Amish microcephaly (MCPHA, OMIM #607196) is a metabolic disorder that has been previously characterized by severe infantile lethal congenital microcephaly and alpha-ketoglutaric aciduria. All reported patients have been from the Pennsylvania Amish community and homozygous for a p.Gly177Ala mutation in SLC25A19. We present a further male patient with MCPHA born to distantly consanguineous parents in Ontario, Canada with Amish ancestors. Microcephaly was evident at 21 weeks gestation on ultrasound. At birth, the facial appearance and brain MRI scan were characteristic of MCPHA, with the additional features of partial agenesis of the corpus callosum and a closed spinal dysraphic state. Urine levels of alpha-ketoglutaric acid were normal at birth and during metabolic crisis, but were markedly elevated during a time of metabolic stability. A severe lactic acidosis was present during metabolic crises and responded to treatment with a high fat diet. At age 7 years, the child is healthy but has severe microcephaly and profound developmental delay. SLC25A19 has been described as a mitochondria inner membrane transporter for both deoxynucleotides and thiamine pyrophosphate (TPP). The biochemical phenotype of MCPHA may be attributable to decreased activity of the three mitochondrial enzymes that require TPP as a cofactor: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched chain amino acid dehydrogenase. We confirm that alpha-ketoglutaric aciduria is not a constant finding in MCPHA and suggest that a persistent lactic acidemia may be more common. The diagnosis should be considered in patients with severe congenital microcephaly, especially in association with lissencephaly, dysgenesis of the corpus callosum, or a spinal dysraphic state.

Small pituitary size in children with Fanconi anemia.

BACKGROUND: Fanconi anemia (FA) is a genetic disorder associated with multiple congenital anomalies, bone marrow failure, and pituitary hypofunction including hypogonadism, thyroid dysfunction, and growth hormone (GH) deficiency. PROCEDURE: Among 44 patients with FA referred to Cincinnati Children's Hospital Medical Center (CCHMC) between 1975 and 2005, 33 had neuroimaging studies, including 11 cranial magnetic resonance imaging (MRIs). Two separate measurements per patient from these MRIs were used to evaluate pituitary height compared to on-site control data of similar measurements of cranial MRIs on 22 age and gender-matched children without any pathology involving the hypothalamic-pituitary system. Growth pattern and endocrine studies were reviewed to assess potential correlation with pituitary size. RESULTS: When compared to the age-gender matched on-site control sample, the mean pituitary height of FA patients was significantly smaller (P < 0.0001; mean +/- SE from mixed effects model with age and gender as covariates: 3.96 +/- 0.32 vs. 5.76 +/- 0.24). Upon further adjusting for the effect of the small head size by including bi-parietal diameter (BPD) as a covariate, the difference remained statistically significant (P = 0.0013). Findings on the growth pattern and endocrinological measurements are as follows: 50% of patients with small pituitary gland were short. GH and adrenal function tests were normal in all tested patients. Thyroid, pubertal status, and glucose regulation were abnormal in 30, 50, and 75% of patients tested. CONCLUSIONS: Children with FA tend to have unsuspected small pituitary glands beyond what is expected from the effects of their stunted growth. Further studies are required to reveal the clinical implications of this finding.

The new syndrome of congenital hypoparathyroidism associated with dysmorphism, growth retardation, and developmental delay--a report of six patients.

We describe six children of Arab origin with a syndrome of congenital hypoparathyroidism, severe growth retardation, developmental delay, and dysmorphism. The most prominent dysmorphic features were microcephaly, facial and dental anomalies, and small hands and feet. Bone age was markedly retarded. Neither immunological nor chromosomal abnormalities were found. Insulin-like growth factor-1 serum levels, measured in two patients, were abnormally low.

Role of cytomegalovirus in inducing congenital anomalies.

Sixty eight infants and newborns were included in the present study being consisted of 55 cases with congenital anomalies and 13 cases of apparently healthy infants acting as controls. Three types of congenital anomaly cases were studied; 17 cases of microcephaly, 17 cases of cerebral palsy and 21 cases with jaundice. All serum samples were tested for cytomegalovirus IgM antibodies (CMV IgM) by microassay ELISA technique to explore the role of CMV infection in inducing congenital anomalies. Six out of the 68 serum samples were found to be positive for CMV IgM. Four of them were detected among the congenital cases (5 our to 55; 7.3%), while 2 cases were among the controls (2 out of 13; 15.4%). The microcephalic group showed 5.9% positivity (one out of 17). The jaundiced infants showed 14.3% positivity (3 out of 21), while all the cerebral palsy cases were negative for CMV IgM. Such a high percentage of CMV IgM in the control group may be attributed to asymptomatic infection, with liability for long term sequelae, particularly hearing loss or ocular abnormalities by 2 years of age. So, continuous follow-up of such asymptomatic cases is essential to control any possible congenital abnormality as early as possible.