Oculocutaneous albinism and bleeding diathesis due to a novel deletion in the HPS3 gene.

Hermansky-Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism (OCA) and bleeding diathesis. To date, 11 HPS types have been reported (HPS-1 to HPS-11), each defined by disease-causing variants in specific genes. Variants in the HPS1 gene were found in approximately 15% of HPS patients, most of whom harbor the Puerto Rican founder mutation. In this study, we report six affected individuals from three nonconsanguineous families of Ashkenazi Jewish descent, who presented with OCA and multiple ecchymoses and had normal platelet number and size. Linkage analysis indicated complete segregation to HPS3. Sequencing of the whole coding region and the intron boundaries of HPS3 revealed a heterozygous c.1163+1G>A variant in all six patients. Long-range PCR amplification revealed that all affected individuals also carry a 14,761bp deletion that includes the 5'UTR and exon 1 of HPS3, encompassing regions with long interspersed nuclear elements. The frequency of the c.1163+1G>A splice site variant was found to be 1:200 in the Ashkenazi Jewish population, whereas the large deletion was not detected in 300 Ashkenazi Jewish controls. These results present a novel HPS3 deletion mutation and suggest that the prevalence of HPS-3 in Ashkenazi Jews is more common than previously thought.

Whole-exome sequencing reveals a monogenic cause in 56% of individuals with laterality disorders and associated congenital heart defects.

BACKGROUND: The molecular basis of heterotaxy and congenital heart malformations associated with disruption of left-right asymmetry is broad and heterogenous, with over 25 genes implicated in its pathogenesis thus far. OBJECTIVE: We sought to elucidate the molecular basis of laterality disorders and associated congenital heart defects in a cohort of 30 unrelated probands of Arab-Muslim descent, using next-generation sequencing techniques. METHODS: Detailed clinical phenotyping followed by whole-exome sequencing (WES) was pursued for each of the probands and their parents (when available). Sanger sequencing was used for segregation analysis of disease-causing mutations in the families. RESULTS: Using WES, we reached a molecular diagnosis for 17 of the 30 probands (56.7%). Genes known to be associated with heterotaxy and/or primary ciliary dyskinesia, in which homozygous pathogenic or likely pathogenic variants were detected, included CFAP53 (CCDC11), CFAP298 (C21orf59), CFAP300, LRRC6, GDF1, DNAAF1, DNAH5, CCDC39, CCDC40, PKD1L1 and TTC25. Additionally, we detected a homozygous disease causing mutation in DAND5, as a novel recessive monogenic cause for heterotaxy in humans. Three additional probands were found to harbour variants of uncertain significance. These included variants in DNAH6, HYDIN, CELSR1 and CFAP46. CONCLUSIONS: Our findings contribute to the current knowledge regarding monogenic causes of heterotaxy and its associated congenital heart defects and underscore the role of next-generation sequencing techniques in the diagnostic workup of such patients, and especially among consanguineous families.

Deep intronic variant in the ARSB gene as the genetic cause for Maroteaux-Lamy syndrome (MPS VI).

Maroteaux-Lamy syndrome (MPS-VI) is a rare autosomal-recessive disorder with a wide spectrum of clinical manifestations, ranging from an attenuated to a rapidly progressive disease. It is caused by variants in ARSB, which encodes the lysosomal arylsulfatase B (ARSB) enzyme, part of the degradation process of glycosaminoglycans in lysosomes. Over 220 variants have been reported so far, with a majority of missense variants. We hereby report two siblings of Bedouin origin with a diagnosis of MPS-VI. Western blots in patient fibroblasts revealed total absence of ARSB protein production. Complete sequencing of the coding region of ARSB did not identify a candidate disease-associated variant. However, deep sequencing of the noncoding region of ARSB by whole genome sequencing (WGS) revealed a c.1142+581A to G variant. The variant is located within intron 5 and fully segregated with the disease in the family. Determination of the genetic cause for these patients enabled targeted treatment by enzyme replacement therapy, along with appropriate genetic counseling and prenatal diagnosis for the family. These results highlight the advantage of WGS as a powerful tool, for improving the diagnostic rate of rare disease-causing variants, and emphasize the importance of studying deep intronic sequence variation as a cause of monogenic disorders.

Four patients with D-bifunctional protein (DBP) deficiency: Expanding the phenotypic spectrum of a highly variable disease.

INTRODUCTION: Peroxisomal D-bifunctional protein (DBP) deficiency is an autosomal recessive disorder historically described as a Zellweger-like syndrome comprising neonatal seizures, retinopathy, hearing loss, dysmorphic features, and other complications. The HSD17B4 gene encodes DBP which is essential for oxidation of peroxisomal substrates. We describe 4 patients - 2 unrelated female girls and 2 monozygotic twin sisters - with DBP deficiency and phenotypic diversity. PATIENT REPORTS: Patient 1 presented neonatally with hypotonia and seizures, and later on developed global developmental delay and regression, sensorineural hearing loss, nystagmus and cortical blindness. The brain MRI demonstrated bilateral peri-sylvian polymicrogyria. Whole exome sequencing revealed 2 mutations in the HSD17B4 gene (c.752G>A, p.(Arg251Gln); c.868 + 1delG).Patient 2 presented with hypotonia, motor delay, and sensorineural hearing loss in infancy, considerable developmental regression during her fourth year, nystagmus, and peripheral neuropathy. Brain MRI demonstrated cerebellar atrophy and abnormal basal ganglia and white matter signal, which appeared after the age of two years. Whole exome sequencing revealed 2 mutations in the HSD17B4 gene (c.14 T>G, p.(Leu5Arg); c.752G>A, p.(Arg251Gln)).Patients 3 and 4, two female monozygotic twins, presented with hypotonia, developmental delay, and macrocephaly from birth, and later on also sensorineural hearing loss, infantile spasms and hypsarrhythmia, and adrenal insufficiency. Brain MRI demonstrated delayed myelination, and an assay of peroxisomal beta oxidation suggested DBP deficiency. Sequencing of the HSD17B4 gene revealed the same 2 mutations as in patient 1. DISCUSSION: We describe 4 patients with variable and diverse clinical picture of DBP deficiency and particularly emphasize the clinical, biochemical, and neuroimaging characteristics. Interestingly, the clinical phenotype varied even between patients with the exact two mutations in the HSD17B4 gene. In addition, in two of the three patients in whom levels of VLCFA including phytanic acid were measured, the levels were within normal limits. This is expanding further the clinical spectrum of this disorder, which should be considered in the differential diagnosis of every patient with hypotonia and developmental delay especially if accompanied by polymicrogyria, seizures, sensorineural hearing loss, or adrenal insufficiency regardless of their VLCFA profile.

Novel homozygous ENPP1 mutation causes generalized arterial calcifications of infancy, thrombocytopenia, and cardiovascular and central nervous system syndrome.

Generalized arterial calcifications of infancy (GACI) is caused by mutations in ENPP1. Other ENPP1-related phenotypes include pseudoxanthoma elasticum, hypophosphatemic rickets, and Cole disease. We studied four children from two Bedouin consanguineous families who presented with severe clinical phenotype including thrombocytopenia, hypoglycemia, hepatic, and neurologic manifestations. Initial working diagnosis included congenital infection; however, patients remained without a definitive diagnosis despite extensive workup. Consequently, we investigated a potential genetic etiology. Whole exome sequencing (WES) was performed for affected children and their parents. Following the identification of a novel mutation in the ENPP1 gene, we characterized this novel multisystemic presentation and revised relevant imaging studies. Using WES, we identified a novel homozygous mutation (c.556G > C; p.Gly186Arg) in ENPP1 which affects a highly conserved protein domain (somatomedin B2). ENPP1-associated genetic diseases exhibit phenotypic heterogeneity depending on mutation type and location. Follow-up clinical characterization of these families allowed us to revise and detect new features of systemic calcifications, which established the diagnosis of GACI, expanding the phenotypic spectrum associated with ENPP1 mutations. Our findings demonstrate that this novel ENPP1 founder mutation can cause a fatal multisystemic phenotype, mimicking severe congenital infection. This also represents the first reported mutation affecting the SMB2 domain, associated with GACI.

Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder.

MECR Mutations Cause Childhood-Onset Dystonia and Optic Atrophy, a Mitochondrial Fatty Acid Synthesis Disorder.

Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285∗), c.247_250del (p.Asn83Hisfs∗4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.

A dominant negative heterozygous G87R mutation in the zinc transporter, ZnT-2 (SLC30A2), results in transient neonatal zinc deficiency.

Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.

McArdle disease: a novel mutation in Jewish families from the Caucasus region.

McArdle disease is caused by a myophosphorylase deficiency consequent to defects in the PYGM gene. A minority of the over-133 known mutations are associated with ethnicity, occurring mainly in patients from western Europe, the United States, and Japan. We identified a novel mutation, c.632delG, in three unrelated families of Jewish descent originating from the Caucasus region. This possibly ethnicity-associated mutation can significantly facilitate the diagnosis in Jews of the Caucasus and contribute to genetic consultations.

The mammalian Nek1 kinase is involved in primary cilium formation.

Recent studies implicate primary cilium (PC) proteins in the etiologies of various polycystic kidney diseases (PKD). NIMA-related kinases (NRKs) are conserved serine/threonine kinases, which are usually defined as 'mitotic kinases'. Murine mutants for the NRKs, nek1 (kat mice) suffer from PKD, suggesting that it may be involved in cilium control. We demonstrated herein that Nek1 is localized to basal body region and that Nek1 overexpression inhibits ciliogenesis in Madin-Darby canine kidney epithelial cells. The number of primary cilia is dramatically reduced in kat2J mouse embryonic fibroblasts culture. It is thus hypothesized that Nek1 links cell cycle progression and the PC cycle.