Loss-of-function variants in JPH1 cause congenital myopathy with prominent facial and ocular involvement.

BACKGROUND: Weakness of facial, ocular and axial muscles is a common clinical presentation in congenital myopathies caused by pathogenic variants in genes encoding triad proteins. Abnormalities in triad structure and function resulting in disturbed excitation-contraction coupling and Ca2+ homeostasis can contribute to disease pathology. METHODS: We analysed exome and genome sequencing data from four unrelated individuals with congenital myopathy characterised by facial, ocular and bulbar involvement. We collected deep phenotypic data from the affected individuals. We analysed the RNA-sequencing (RNA-seq) data of F3-II.1 and performed gene expression outlier analysis in 129 samples. RESULTS: The four probands had a remarkably similar clinical presentation with prominent facial, ocular and bulbar features. Disease onset was in the neonatal period with hypotonia, poor feeding, cleft palate and talipes. Muscle weakness was generalised but prominent in the lower limbs with facial weakness also present. All patients had myopathic facies, bilateral ptosis, ophthalmoplegia and fatigability. Muscle biopsy on light microscopy showed type 1 myofiber predominance and ultrastructural analysis revealed slightly reduced triads, and structurally abnormal sarcoplasmic reticulum.DNA sequencing identified four unique homozygous loss-of-function variants in JPH1, encoding junctophilin-1 in the four families; one stop-gain (c.354C>A;p.Tyr118*) and three frameshift (c.373delG;p.Asp125Thrfs*30, c.1738delC;p.Leu580Trpfs*16 and c.1510delG;p. Glu504Serfs*3) variants. Muscle RNA-seq showed strong downregulation of JPH1 in the F3 proband. CONCLUSIONS: Junctophilin-1 is critical for the formation of skeletal muscle triad junctions by connecting the sarcoplasmic reticulum and T-tubules. Our findings suggest that loss of JPH1 results in a congenital myopathy with prominent facial, bulbar and ocular involvement.

Biallelic variants in SLC38A3 encoding a glutamine transporter cause epileptic encephalopathy.

The solute carrier (SLC) superfamily encompasses >400 transmembrane transporters involved in the exchange of amino acids, nutrients, ions, metals, neurotransmitters and metabolites across biological membranes. SLCs are highly expressed in the mammalian brain; defects in nearly 100 unique SLC-encoding genes (OMIM: https://www.omim.org) are associated with rare Mendelian disorders including developmental and epileptic encephalopathy and severe neurodevelopmental disorders. Exome sequencing and family-based rare variant analyses on a cohort with neurodevelopmental disorders identified two siblings with developmental and epileptic encephalopathy and a shared deleterious homozygous splicing variant in SLC38A3. The gene encodes SNAT3, a sodium-coupled neutral amino acid transporter and a principal transporter of the amino acids asparagine, histidine, and glutamine, the latter being the precursor for the neurotransmitters GABA and glutamate. Additional subjects with a similar developmental and epileptic encephalopathy phenotype and biallelic predicted-damaging SLC38A3 variants were ascertained through GeneMatcher and collaborations with research and clinical molecular diagnostic laboratories. Untargeted metabolomic analysis was performed to identify novel metabolic biomarkers. Ten individuals from seven unrelated families from six different countries with deleterious biallelic variants in SLC38A3 were identified. Global developmental delay, intellectual disability, hypotonia, and absent speech were common features while microcephaly, epilepsy, and visual impairment were present in the majority. Epilepsy was drug-resistant in half. Metabolomic analysis revealed perturbations of glutamate, histidine, and nitrogen metabolism in plasma, urine, and CSF of selected subjects, potentially representing biomarkers of disease. Our data support the contention that SLC38A3 is a novel disease gene for developmental and epileptic encephalopathy and illuminate the likely pathophysiology of the disease as perturbations in glutamine homeostasis.

Mutations in phospholipase C eta-1 (PLCH1) are associated with holoprosencephaly.

BACKGROUND: Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities. METHODS: We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (PLCH1). Immunocytochemistry was used to examine the expression pattern of PLCH1 in human embryos. We used SHH as a marker of developmental stage and of early embryonic anatomy. RESULTS: In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygous PLCH1 c.2065C>T, p.(Arg689*) variant. In the second family, two siblings had alobar holoprosencephaly and cyclopia with a homozygous PLCH1 c.4235delA, p.(Cys1079ValfsTer16) variant. All parents were healthy carriers, with no holoprosencephaly spectrum features. We found that the subcellular localisation of PLCH1 is cytoplasmic, but the p.(Cys1079ValfsTer16) variant was predominantly nuclear. Human embryo immunohistochemistry showed PLCH1 to be expressed in the notorcord, developing spinal cord (in a ventral to dorsal gradient), dorsal root ganglia, cerebellum and dermatomyosome, all tissues producing or responding to SHH. Furthermore, the embryonic subcellular localisation of PLCH1 was exclusively cytoplasmic, supporting protein mislocalisation contributing to the pathogenicity of the p.(Cys1079ValfsTer16) variant. CONCLUSION: Our data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype.

Biallelic ZNFX1 variants are associated with a spectrum of immuno-hematological abnormalities.

Biallelic changes in the ZNFX1 gene have been recently reported to cause severe familial immunodeficiency. Through a search of our bio/databank with information from genetic testing of >55 000 individuals, we identified nine additional patients from seven families with six novel homozygous ZNFX1 variants. Consistent with the previously described phenotype, our patients suffered from monocytosis, thrombocytopenia, hepatosplenomegaly, recurrent infections, and lymphadenopathy. The two most severely affected probands also had renal involvement and clinical presentations compatible with hemophagocytic lymphohistiocytosis. The disease was less lethal among our patients than previously reported. We identified two missense changes, two variants predicted to result in complete protein loss through nonsense-mediated decay, and two frameshift changes that likely introduce a truncation. Our findings (i) independently confirm the role of ZNFX1 in primary genetic immunodeficiency, (ii) expand the genetic and clinical spectrum of ZNFX1-related disease, and (iii) illustrate the utility of large, well-curated, and continually updated genotype-phenotype databases in resolving molecular diagnoses of patients with initially negative genetic testing findings.

The genotypic and phenotypic spectrum of pycnodysostosis in Saudi Arabia: Novel variants and clinical findings.

Pycnodysostosis is characterized by short stature, osteosclerosis, acro-osteolysis, increased tendency of fractures, and distinctive dysmorphic features. It is a rare autosomal recessive disease caused by biallelic CTSK mutations. The clinical details of 18 patients from Saudi Arabia were reviewed. Short stature, osteopetrosis, acro-osteolysis, and distinctive facial dysmorphism were documented in all cases. Our results highlight the significant complications associated with this disease. The large anterior fontanelle is one of the cardinal signs of this disease; however, half of our patients had small fontanelles and a quarter had craniosynostosis, which caused optic nerve compression. Sleep apnea was of the major complications in three patients. Bone fracture can be a presenting symptom, and in our patients it mainly occurred after the age of 3 years. Bone marrow suppression was seen in a single patient of our cohort who was misdiagnosed initially with malignant osteopetrosis. In this study, we also describe two novel (c.5G > A [p.Trp2Ter], c.538G > A [p.Gly180Ser]) and two reported (c.244-29 A > G, c.830C > T [p.Ala277Val]) CTSK mutations. Our results indicate that the recurrent intronic variant, c.244-29 A > G is likely to be a founder mutation, as it was found in 78% (14/18 patients) of our cohort belonging to the same tribe.

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.

Epilepsy, neuropsychiatric phenotypes, neuroimaging findings, and genotype-neurophenotype correlation in 22q11.2 deletion syndrome.

OBJECTIVE: To describe the epilepsy, neuropsychiatric manifestations, and neuroimaging findings in a group of patients with 22q11.2 DS, and to correlate the size of the deleted genetic material with the severity of the phenotype. METHODS: We retrospectively analyzed the medical records of 28 patients (21 pediatric patients and 7 adults) with a genetically confirmed diagnosis of 22q11.2 DS. Clinical data (epilepsy, neurological exam, neuropsychological and developmental assessment, and psychiatric disorders), neuroimaging, and cytogenetic tests were analyzed RESULTS: Of the 28 patients with 22q11.2 DS, 6 (21.4%) had epileptic seizures, 2 had symptomatic hypocalcemic seizures, 4 (14.2%) had a psychiatric disorder, which comprised of attention deficit hyperactivity disorder, autism spectrum disorder, psychosis, and mood disorder, and 17 (60.7%) had developmental delay. All patients with epilepsy had a developmental delay. Twelve patients underwent a neuropsychology assessment. Intellectual levels ranged from moderate intellectual disability (7/12, 58%) to average (5/12, 41.6%). Of the 16 patients, 6 (37.5%) had a normal brain, while 10 (62.5%) had abnormal neuroimaging findings. No significant correlation was found between the size of the deleted genetic material and the severity of the phenotype. CONCLUSION: 22q11.2DS patients are at high risk to develop epilepsy, neuropsychiatric manifestations, and structural brain abnormalities. This indicates that this defined genetic locus is crucial for the development of the nervous system, and patients with 22q11.2 DS have genetic susceptibility to develop epilepsy.

Neurological expression of an inherited translocation of chromosomal 1 and 7.

An unbalanced translocation of chromosome 1 and 7 (t[1;7]) associated with neurological phenotype and brain malformation has rarely been reported. This clinical report describes 3 siblings with brain malformations and a 13.5 Mb duplication of 1q42.3q44, and a 7.6 Mb duplication of 7q36.1q36.3 detected by array comparative genomic hybridization. This unbalanced t(1;7) was found to be inherited from a balanced translocation from the mother. All the patients presented with hypotonia, microcephaly, developmental delay, seizures, abnormal corpus callosum and abnormal cerebellum.

Crisponi/CISS1 syndrome: A case series.

Crisponi/CISS1 syndrome (MIM#272430) is a rare autosomal recessive disease characterized by major feeding difficulties, camptodactyly, and anhidrosis in early childhood; and the subsequent development of paradoxical cold-induced sweating and scoliosis later in life. The syndrome is caused by biallelic mutations in CRLF1 or, much less commonly, CLCF1. Although genotype/phenotype correlation has been elusive, it has been suggested that the level of the mutant protein may correlate with the phenotypic severity. However, we show in this series of 12 patients from four families, all previously unpublished, that the homogeneity of the recently described c.983dupG (p.Ser328Argfs∗2) mutation in CRLF1 was associated with a highly variable degree of severity, and that the phenotype significantly overlaps with the recently described COG6-related anhidrosis syndrome (MIM#615328). Another fifth previously unpublished family is also described with a novel mutation in CRLF1, c.605delC (p.Ala202Valfs*32). In Saudi Arabia the prevalence of the syndrome is probably underestimated due to the difficulty in making the diagnosis considering the complex phenotype with typical neonatal and evolutive features.

Effect of consanguinity on birth defects in Saudi women: results from a nested case-control study.

BACKGROUND: The role of consanguinity in the etiology of structural birth defects outside of chromosomal and inherited disorders has always been debated. We studied the independent role of consanguinity on birth defects in Saudi women with a high prevalence of consanguineous marriages. METHODS: This case and control study was nested within a 3-year prospective cohort study to examine patterns of fetal and neonatal malformations in Saudi women at Prince Sultan Military Medical City (PSMMC), Riyadh -Saudi Arabia. Consanguineous marriages were defined as marriages with first or second cousins (related); unions beyond second cousins (distant relatives) were considered unrelated for this study. RESULTS: During the 3-year study (July 2010 through June 2013), there were 28,646 total births; of these, we included 1,179 babies with major birth defects, and 1,262 babies as their controls. The consanguinity prevalence for all included women was 49.6%. The consanguinity among babies with major Birth Defects (BDs) was 54.5% and 45.2% for controls (P < 0.0002). The prevalence of major birth defects was 41.1 per 1000 total births. Univariate analysis showed that consanguinity had a statistically significant contribution in babies born with genetic syndromes, isolated renal defects, and isolated other defects (P < 0.05). Multivariate logistic regression analyses showed that consanguinity was an independent risk factor for this high prevalence of birth defects in the study population (P < 0.0002). CONCLUSION: The prevalence of major birth defects in the study population is higher than what is reported from European countries. Consanguinity is a significant independent risk factor for the high prevalence of birth defects.

Intellectual Disability in Two Brothers Caused by De Novo Novel Unbalanced Translocation (13;18) (q34,q23) and De Novo Microdeletion 6q25 Syndrome.

We report here two brothers with an intellectual disability (ID), dysmorphic features, speech delay, and congenital hypotonia, with chromosomal microarray confirmed. However, two different de novo chromosomal aberrations; unbalanced translocations (13;18) (q34,q23) were found in the elder boys and de novo 6q25 deletion in the second boy. The boy with 13q34 microdeletion and 18q23 microduplication suffered from ID, obesity, dysmorphic features, speech delay, and seizure while the one with 6q25 deletion presented with ID and speech delay. Both parents were tested and were normal. The third child had mild hypotonia at infancy, which improved later. Whole-exome sequencing (WES) showed the three boys carried a likely benign variant in MED12, inherited from the healthy, asymptomatic mother. The father suffered from rheumatoid arthritis and was on chemotherapy during the conception of the first two affected boys. This report places emphasis on the use of a chromosomal microarray in patients with ID, even with familial cases, and reports the paternal use of methotrexate.

Peripheral venous route for administration of ammonul infusion for treatment of acute hyperammonemia. An experience from a tertiary center in Saudi Arabia.

OBJECTIVES: To determine the local effects of peripheral Ammonul infusion on the skin and the subcutaneous tissues.  Methods: This retrospective study was conducted at Prince Sultan Military Medical City, Riyadh, Saudi Arabia. All children less than 16 years of age admitted between December 2015 and October 2018 with hyperammonemia and received Ammonul infusion for treatment were recruited. Results: Twenty-one patients received the Ammonul infusion. They were admitted 58 times with acute hyperammonemia during the study period, with an average of 2.8 admissions per patient. The mean age of the included patients was 49.5 months. The most frequent underlying diagnoses were propionic acidemia (n=9), urea cycle disorders (n=5), and intrinsic liver disease (n=3). All participants received Ammonul through peripheral lines except 3 who received it through central lines. No extravasation, burns, or other local side effects were observed in this cohort. CONCLUSION: This data indicate that the use of Ammonul through a peripheral venous route appears to be safe and not associated with infusion-related local adverse effects.

Fructose-1,6-bisphosphatase deficiency with confirmed molecular diagnosis. An important cause of hypoglycemia in children.

OBJECTIVES: To draw attention towards fructose-1,6-bisphosphatase (FBPase) deficiency as an important cause of hypoglycemia and lactic acidosis and to implement preventive strategies. Methods: This observational, cross-sectional study was conducted on 7 Saudi patients with genetically confirmed FBPase deficiency from 2008 to 2018 at Prince Sultan Military Medical City, Riyadh, Saudi Arabia. Results: Participants ranged in age from 1-10 years, and all presented with recurrent hypoglycemia. All but one had associated severe metabolic acidosis, and 3 patients (42.9%) presented with hypoglycemia and severe acidosis since birth. The mean duration from presentation to diagnosis was 39.4 months, as other diagnoses, like glycogen storage diseases and mitochondrial diseases needed to be ruled out. Development was normal apart from speech delay in one patient with a novel variant of the FBP1 gene. All patients have homozygous variants in the FBP1 gene.  Conclusion: Fructose-1,6-bisphosphatase is an important cause of hypoglycemia and acidosis; therefore, it is important to offer early molecular diagnostics in any child presenting with these symptoms. Molecular diagnostics should always be undertaken to confirm the diagnosis and for further preventive strategies.

Incidence of newborn screening disorders among 56632 infants in Central Saudi Arabia. A 6-year study.

OBJECTIVES: To determine the incidence of newborn screening (NBS) disorders and to study the key performance indicators of the program. METHODS: This retrospective single-center study enrolled all infants who underwent NBS from January 2012 to December 2017 at Prince Sultan Military Medical City, Riyadh, Saudi Arabia. We screened 17 NBS disorders. Blood samples were collected 24 hours after birth. If the initial result was positive, a second sample was collected. True positive cases were immediately referred for medical management. Data were extracted from laboratory computerized and non-computerized records using case report forms. RESULTS: During the study period, 56632 infants underwent NBS with a coverage rate of 100%. Thirty-eight cases were confirmed. The incidence of congenital hypothyroidism was 1:3775. The positive predictive value for the detection of congenital hypothyroidism was 11.8%. Propionic aciduria was the most common metabolic disorder, with an incidence of 1:14158. Very long-chain acyl CoA dehydrogenase deficiency and glutaric aciduria type 1 had an incidence of 1:18877 each. Phenylketonuria, biotinidase deficiency, maple syrup urine disease, and citrullinemia had an incidence of 1:28316 each. However, galactosemia and 3-methyl crotonyl carboxylase deficiency had the lowest incidence of 1:56632. CONCLUSION: The NBS coverage rate at our facility was 100%. Congenital hypothyroidism was the most frequently detected disorder with an incidence that matches worldwide figures. The incidence of other inherited disorders was consistent with regional figures.

Congenital anomalies and associated risk factors in a Saudi population: a cohort study from pregnancy to age 2 years.

OBJECTIVE: To assess the three key issues for congenital anomalies (CAs) prevention and care, namely, CA prevalence, risk factor prevalence and survival, in a longitudinal cohort in Riyadh, Saudi Arabia. SETTING: Tertiary care centre, Riyadh, Saudi Arabia. PARTICIPANTS: Saudi women enrolled during pregnancy over 3 years and their 28 646 eligible pregnancy outcomes (births, stillbirths and elective terminations of pregnancy for foetal anomalies). The nested case-control study evaluated the CA risk factor profile of the underlying cohort. All CA cases (1179) and unaffected controls (1262) were followed through age 2 years. Referred mothers because of foetal anomaly and mothers who delivered outside the study centre and their pregnancy outcome were excluded. PRIMARY OUTCOME MEASURES: Prevalence and pattern of major CAs, frequency of CA-related risk factors and survival through age 2 years. RESULTS: The birth prevalence of CAs was 412/10 000 births (95% CI 388.6 to 434.9), driven mainly by congenital heart disease (148 per 10 000) (95% CI 134 to 162), renal malformations (113, 95% CI 110 to 125), neural tube defects (19, 95% CI 25.3 to 38.3) and chromosomal anomalies (27, 95% CI 21 to 33). In this study, the burden of potentially modifiable risk factors included high rates of diabetes (7.3%, OR 1.98, 95% CI 1.04 to 2.12), maternal age >40 years (7.0%, OR 2.1, 95% CI 1.35 to 3.3), consanguinity (54.5%, OR 1.5, 95% CI 1.28 to 1.81). The mortality for live births with CAs at 2 years of age was 15.8%. CONCLUSIONS: This study documented specific opportunities to improve primary prevention and care. Specifically, folic acid fortification (the neural tube defect prevalence was >3 times that theoretically achievable by optimal fortification), preconception diabetes screening and consanguinity-related counselling could have significant and broad health benefits in this cohort and arguably in the larger Saudi population.

Genetic, clinical and biochemical characterization of a large cohort of patients with hyaline fibromatosis syndrome.

BACKGROUND: Hyaline fibromatosis syndrome (HFS) is a rare clinical condition in which bi-allelic variants in ANTXR2 are associated with extracellular hyaline deposits. It manifests as multiple skin nodules, patchy hyperpigmentation, joint contractures and severe pain with movement. HFS shows some clinical overlap to Farber disease (FD), a recessive lysosomal storage disorder. RESULTS: We here present the largest cohort of independent, genetically confirmed HFS cases reported to date: in 19 unrelated index patients, we identified ten distinct homozygous ANTXR2 mutations, three of which are novel frame-shift variants. The associated clinical data are consistent with the previous hypothesis of non-truncating variants in the terminal exons 13-17 to confer rather mild phenotypes. The novel observation of gender-dependent disease manifestation in our cohort received support from a meta-analysis of all previously published cases. Untargeted blood-based metabolomics revealed patient samples to be biochemically distinct from control samples. Numerous potential HFS biomarker metabolites could thus be identified. We also found metabolomics profiles of HFS patients to highly overlap with those from FD patients. CONCLUSIONS: Our study extends the mutational spectrum for HFS, suggests gender-dependency of manifestation, and provides pilot metabolomics data for biomarker identification and a better pathomechanistic understanding of the disorder.