Bi-allelic ACBD6 variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders.

The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Using exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with myristic acid alkyne (YnMyr) chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), aged 1-50 years, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%) and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%) and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%) and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each) as well as hypertrophy of the clava (24%) were common neuroimaging findings. Acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localization and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-myristoylation was similarly affected in acbd6-deficient zebrafish and X. tropicalis models, including Fus, Marcks and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders.

Recurrent homozygous damaging mutation in TMX2, encoding a protein disulfide isomerase, in four families with microlissencephaly.

BACKGROUND: Protein disulfide isomerase (PDI) proteins are part of the thioredoxin protein superfamily. PDIs are involved in the formation and rearrangement of disulfide bonds between cysteine residues during protein folding in the endoplasmic reticulum and are implicated in stress response pathways. METHODS: Eight children from four consanguineous families residing in distinct geographies within the Middle East and Central Asia were recruited for study. All probands showed structurally similar microcephaly with lissencephaly (microlissencephaly) brain malformations. DNA samples from each family underwent whole exome sequencing, assessment for repeat expansions and confirmatory segregation analysis. RESULTS: An identical homozygous variant in TMX2 (c.500G>A), encoding thioredoxin-related transmembrane protein 2, segregated with disease in all four families. This variant changed the last coding base of exon 6, and impacted mRNA stability. All patients presented with microlissencephaly, global developmental delay, intellectual disability and epilepsy. While TMX2 is an activator of cellular C9ORF72 repeat expansion toxicity, patients showed no evidence of C9ORF72 repeat expansions. CONCLUSION: The TMX2 c.500G>A allele associates with recessive microlissencephaly, and patients show no evidence of C9ORF72 expansions. TMX2 is the first PDI implicated in a recessive disease, suggesting a protein isomerisation defect in microlissencephaly.

A novel biallelic single base insertion in WNK1 in a Pakistani family with congenital insensitivity to pain.

Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare, recessively inherited neurological condition frequently involving insensitivity to pain. The subtype, HSAN2A, results from mutations in the gene WNK1. We identified a consanguineous Pakistani family with three affecteds showing symptoms of HSANII. We performed microarray genotyping, followed by homozygosity-by-descent (HBD) mapping, which indicated several significant HBD regions, including ~6 Mb towards the terminus of chromosome 12p, spanning WNK1. Simultaneously, we performed whole exome sequencing (WES) on one of the affected brothers, and identified a homozygous 1 bp insertion variant, Chr12:978101dupA, within exon 10. This variant, confirmed to segregate in the family, is predicted to truncate the protein (NM_213655.4:c.3464delinsAC; p.(Thr1155Asnfs*11) and lead to nonsense-mediated mRNA decay of the transcript. Previous studies of congenital pain insensitivity/HSANII in Pakistani families have identified mutations in SCN9A. Our study identified a previously unreported WNK1 mutation segregating with congenital pain insensitivity/HSANII in a Pakistani family.

Mutations in MBOAT7, Encoding Lysophosphatidylinositol Acyltransferase I, Lead to Intellectual Disability Accompanied by Epilepsy and Autistic Features.

The risk of epilepsy among individuals with intellectual disability (ID) is approximately ten times that of the general population. From a cohort of >5,000 families affected by neurodevelopmental disorders, we identified six consanguineous families harboring homozygous inactivating variants in MBOAT7, encoding lysophosphatidylinositol acyltransferase (LPIAT1). Subjects presented with ID frequently accompanied by epilepsy and autistic features. LPIAT1 is a membrane-bound phospholipid-remodeling enzyme that transfers arachidonic acid (AA) to lysophosphatidylinositol to produce AA-containing phosphatidylinositol. This study suggests a role for AA-containing phosphatidylinositols in the development of ID accompanied by epilepsy and autistic features.

Risk of meningomyelocele mediated by the common 22q11.2 deletion.

Meningomyelocele is one of the most severe forms of neural tube defects (NTDs) and the most frequent structural birth defect of the central nervous system. We assembled the Spina Bifida Sequencing Consortium to identify causes. Exome and genome sequencing of 715 parent-offspring trios identified six patients with chromosomal 22q11.2 deletions, suggesting a 23-fold increased risk compared with the general population. Furthermore, analysis of a separate 22q11.2 deletion cohort suggested a 12- to 15-fold increased NTD risk of meningomyelocele. The loss of Crkl, one of several neural tube-expressed genes within the minimal deletion interval, was sufficient to replicate NTDs in mice, where both penetrance and expressivity were exacerbated by maternal folate deficiency. Thus, the common 22q11.2 deletion confers substantial meningomyelocele risk, which is partially alleviated by folate supplementation.

Identification of mutations in TRAPPC9, which encodes the NIK- and IKK-beta-binding protein, in nonsyndromic autosomal-recessive mental retardation.

Mental retardation/intellectual disability is a devastating neurodevelopmental disorder with serious impact on affected individuals and their families, as well as on health and social services. It occurs with a prevalence of approximately 2%, is an etiologically heterogeneous condition, and is frequently the result of genetic aberrations. Autosomal-recessive forms of nonsyndromic MR (NS-ARMR) are believed to be common, yet only five genes have been identified. We have used homozygosity mapping to search for the gene responsible for NS-ARMR in a large Pakistani pedigree. Using Affymetrix 5.0 single nucleotide polymorphism (SNP) microarrays, we identified a 3.2 Mb region on 8q24 with a continuous run of 606 homozygous SNPs shared among all affected members of the family. Additional genotype data from microsatellite markers verified this, allowing us to calculate a two-point LOD score of 5.18. Within this region, we identified a truncating homozygous mutation, R475X, in exon 7 of the gene TRAPPC9. In a second large NS-ARMR/ID family, previously linked to 8q24 in a study of Iranian families, we identified a 4 bp deletion within exon 14 of TRAPPC9, also segregating with the phenotype and truncating the protein. This gene encodes NIK- and IKK-beta-binding protein (NIBP), which is involved in the NF-kappaB signaling pathway and directly interacts with IKK-beta and MAP3K14. Brain magnetic resonance imaging of affected individuals indicates the presence of mild cerebral white matter hypoplasia. Microcephaly is present in some but not all affected individuals. Thus, to our knowledge, this is the sixth gene for NS-ARMR to be discovered.

5 versus 10 days of treatment with ceftriaxone for bacterial meningitis in children: a double-blind randomised equivalence study.

BACKGROUND: Bacterial meningitis is an important cause of morbidity and mortality in developing countries, but the duration of treatment is not well established. We aimed to compare the efficacy of 5 and 10 days of parenteral ceftriaxone for the treatment of bacterial meningitis in children. METHODS: We did a multicountry, double-blind, placebo-controlled, randomised equivalence study of 5 versus 10 days of treatment with ceftriaxone in children aged 2 months to 12 years with purulent meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae type B, or Neisseria meningitidis. Our study was done in ten paediatric referral hospitals in Bangladesh, Egypt, Malawi, Pakistan, and Vietnam. We randomly assigned children who were stable after 5 days of treatment, through site-balanced computer-generated allocation lists, to receive a further 5 days of ceftriaxone or placebo. Patients, their guardians, and staff were masked to study-group allocation. Our primary outcomes were bacteriological failure or relapse. Our analysis was per protocol. This study is registered with the International Standard Randomised Controlled Trial Number Register, number ISRCTN38717320. FINDINGS: We included 1004 of 1027 children randomly assigned to study groups in our analyses; 496 received treatment with ceftriaxone for 5 days, and 508 for 10 days. In the 5-day treatment group, two children (one infected with HIV) had a relapse; there were no relapses in the 10-day treatment group and there were no bacteriological failures in either study group. Side-effects of antibiotic treatment were minor and similar in both groups. INTERPRETATION: In children beyond the neonatal age-group with purulent meningitis caused by S pneumoniae, H influenzae type b, or N meningitidis who are stable by day 5 of ceftriaxone treatment, the antibiotic can be safely discontinued. FUNDING: United States Agency for International Development.

CC2D2A, encoding a coiled-coil and C2 domain protein, causes autosomal-recessive mental retardation with retinitis pigmentosa.

Autosomal-recessive inheritance is believed to be relatively common in mental retardation (MR), although only four genes for nonsyndromic autosomal-recessive mental retardation (ARMR) have been reported. In this study, we ascertained a consanguineous Pakistani family with ARMR in four living individuals from three branches of the family, plus an additional affected individual later identified as a phenocopy. Retinitis pigmentosa was present in affected individuals, but no other features suggestive of a syndromic form of MR were found. We used Affymetrix 500K microarrays to perform homozygosity mapping and identified a homozygous and haploidentical region of 11.2 Mb on chromosome 4p15.33-p15.2. Linkage analysis across this region produced a maximum two-point LOD score of 3.59. We sequenced genes within the critical region and identified a homozygous splice-site mutation segregating in the family, within a coiled-coil and C2 domain-containing gene, CC2D2A. This mutation leads to the skipping of exon 19, resulting in a frameshift and a truncated protein lacking the C2 domain. Conservation analysis for CC2D2A suggests a functional domain near the C terminus as well as the C2 domain. Preliminary functional studies of CC2D2A suggest a possible role in Ca(2+)-dependent signal transduction. Identifying the function of CC2D2A, and a possible common pathway with CC2D1A, in correct neuronal development and functioning may help identify possible therapeutic targets for MR.

Use of ACTH and prednisolone in infantile spasms: experience from a developing country.

BACKGROUND: Adrenocorticotrophic hormone (ACTH) and prednisone are both used to treat infantile spasms (IS) in West syndrome. In many countries, ACTH is expensive and difficult to obtain whereas, prednisone or prednisolone are cheap, given orally and easily available. AIMS: The purpose of this retrospective data analysis was to compare the efficacy and cost of ACTH and prednisolone in the treatment of IS from the perspective of a developing country. METHODS: Patients admitted with West syndrome in Children's Hospital, Islamabad, between January 1995 and December 2001 were included in the analysis. The diagnosis was made after eliciting a history of characteristic seizures and detecting hypsarrhythmia on the EEG. Parents were offered the use of either ACTH administered by intramuscular injection or prednisolone given orally. ACTH was expensive and difficult to obtain whereas prednisolone was cheap and easily available. RESULTS: One hundred and five children were included in the study. Sixty-three were boys and their age ranged from 2 months to 3 years with a mean of 11 months. Thirty-three children received ACTH injections; 27 showed improvement and 11 remained spasms free after discontinuation of injections. Seventy-two patients were given oral prednisolone, 51 responded and 17 remained spasms free after oral steroids were stopped. Overall outcome was similar in both groups. The cost of ACTH injection was more than 100 times the cost of oral prednisolone. CONCLUSION: No significant difference was seen in the final outcome in both treatment groups. Since prednisolone is inexpensive, easily available and given orally, it is the preferred mode of therapy.

Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies.

Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction.

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.

Intracranial tuberculomas and caries spine: an experience from Children's Hospital Islamabad.

BACKGROUND: Pulmonary tuberculosis in infants and children in Pakistan is quite common. However, there is limited data about uncommon forms of tuberculous infection of central nervous system. Tuberculosis of nervous system is a serious disease and if not treated adequately, carries high morbidity and mortality. This study was undertaken to highlight the occurrence of relatively uncommon forms of tuberculosis in children. METHODS: This was a retrospective review of the case records of the patients who were admitted in Children's Hospital, Islamabad with the diagnosis of cerebral tubercloma or tuberculosis of spine between January, 1994 and August, 2002. Diagnosis was based on clinical features, history of contact with tuberculous patient, abnormalities on chest X-rays and neuroimaging studies of brain and spine. RESULTS: Nine children had cerebral tuberculomas, six more than one and in five were bilateral. Eight had caries spine, 2 cervical, 4 thoracic and 2 lumbar regions. Three had been vaccinated with Bacillus-Calemette-Guerin and family history of tuberculosis was positive in 7 (41%) children. Nine (53 %) patients had evidence of pulmonary tuberculosis on chest x-ray. Ten computed scan brain, four magnetic resonance imaging, two spinal scan and two myelographic studies were performed. Three patients with tuberculomas and five with caries recovered and 8 were left with neurological deficit. One child with cerebral tuberculomas died. CONCLUSION: Intracranial tuberculomas and spinal tuberculosis are not rare problems in children. Early diagnosis and prompt antituberculous therapy are the most important factors for the favourable outcome.

Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders.

Hereditary spastic paraplegias (HSPs) are neurodegenerative motor neuron diseases characterized by progressive age-dependent loss of corticospinal motor tract function. Although the genetic basis is partly understood, only a fraction of cases can receive a genetic diagnosis, and a global view of HSP is lacking. By using whole-exome sequencing in combination with network analysis, we identified 18 previously unknown putative HSP genes and validated nearly all of these genes functionally or genetically. The pathways highlighted by these mutations link HSP to cellular transport, nucleotide metabolism, and synapse and axon development. Network analysis revealed a host of further candidate genes, of which three were mutated in our cohort. Our analysis links HSP to other neurodegenerative disorders and can facilitate gene discovery and mechanistic understanding of disease.

Exome sequencing can improve diagnosis and alter patient management.

The translation of "next-generation" sequencing directly to the clinic is still being assessed but has the potential for genetic diseases to reduce costs, advance accuracy, and point to unsuspected yet treatable conditions. To study its capability in the clinic, we performed whole-exome sequencing in 118 probands with a diagnosis of a pediatric-onset neurodevelopmental disease in which most known causes had been excluded. Twenty-two genes not previously identified as disease-causing were identified in this study (19% of cohort), further establishing exome sequencing as a useful tool for gene discovery. New genes identified included EXOC8 in Joubert syndrome and GFM2 in a patient with microcephaly, simplified gyral pattern, and insulin-dependent diabetes. Exome sequencing uncovered 10 probands (8% of cohort) with mutations in genes known to cause a disease different from the initial diagnosis. Upon further medical evaluation, these mutations were found to account for each proband's disease, leading to a change in diagnosis, some of which led to changes in patient management. Our data provide proof of principle that genomic strategies are useful in clarifying diagnosis in a proportion of patients with neurodevelopmental disorders.

Piracetam in severe breath holding spells.

BACKGROUND: Breath holding spells (BHS) are apparently frightening events occurring in otherwise healthy children. Generally, no medical treatment is recommended and parental reassurance is believed to be enough, however, severe BHS can be very stressful for the parents and a pharmacological agent may be desired in some of these children. OBJECTIVE: In this prospective study aim was to determine the usefulness of piracetam as prophylactic treatment for severe BHS. METHODS: Children were recruited from Neurology Clinic in Children's Hospital, Islamabad between January 2002 to December 2004. Diagnosis of BHS was based on characteristic history and normal physical examination. Piracetam was prescribed to those children who were diagnosed as severe BHS in a dose ranging from 50-100 mg/kg/day. Iron supplements were added if hemoglobin was less than 10 gm%. Patients were seen at 2-4 weeks interval and follow-up was continued until 3 months after the cessation of drug therapy. RESULTS: Fifty-two children were enrolled in the study, 34 boys and 18 girls. Ages ranged from 4 weeks to 5 years with mean age of 17 months. In 81% of children, spells disappeared completely and in 9% frequency was reduced to less than one per month and of much lesser intensity. Prophylaxis was given for 3-6 months (mean 5) duration. CONCLUSIONS: Piracetam is an effective prophylactic treatment for severe BHS.