A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects.

Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.

Identification and characterization of Aurora kinase B and C variants associated with maternal aneuploidy.

STUDY QUESTION: Are single nucleotide variants (SNVs) in Aurora kinases B and C (AURKB, AURKC) associated with risk of aneuploid conception? SUMMARY ANSWER: Two SNVs were found in patients with extreme aneuploid concepti rates with respect to their age; one variant, AURKC p.I79V, is benign, while another, AURKB p.L39P, is a potential gain-of-function mutant with increased efficiency in promoting chromosome alignment. WHAT IS KNOWN ALREADY: Maternal age does not always predict aneuploidy risk, and rare gene variants can be drivers of disease. The AURKB and AURKC regulate chromosome segregation, and are associated with reproductive impairments in mouse and human. STUDY DESIGN, SIZE, DURATION: An extreme phenotype sample selection scheme was performed for variant discovery. Ninety-six DNA samples were from young patients with higher than average embryonic aneuploidy rates and an additional 96 DNA samples were from older patients with lower than average aneuploidy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using the192 DNA samples, the coding regions of AURKB and AURKC were sequenced using next generation sequencing. To assess biological significance, we expressed complementary RNA encoding the human variants in mouse oocytes. Assays such as determining subcellular localization and assessing catalytic activity were performed to determine alterations in protein function during meiosis. MAIN RESULTS AND THE ROLE OF CHANCE: Ten SNVs were identified using three independent variant-calling methods. Two of the SNVs (AURKB p.L39P and AURKC p.I79V) were non-synonymous and identified by at least two variant-identification methods. The variant encoding AURKC p.I79V, identified in a young woman with a higher than average rate of aneuploid embryos, showed wild-type localization pattern and catalytic activity. On the other hand, the variant encoding AURKB p.L39P, identified in an older woman with lower than average rates of aneuploid embryos, increased the protein's ability to regulate alignment of chromosomes at the metaphase plate. These experiments were repeated three independent times using 2-3 mice for each trial. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Biological significance of the human variants was assessed in an in vitro mouse oocyte model where the variants are over-expressed. Therefore, the human protein may not function identically to the mouse homolog, or the same in mouse oocytes as in human oocytes. Furthermore, supraphysiological expression levels may not accurately reflect endogenous activity. Moreover, the evaluated variants were identified in one patient each, and no trial linking the SNV to pregnancy outcomes was conducted. Finally, the patient aneuploidy rates were established by performing comprehensive chromosome screening in blastocysts, and because of the link between female gamete aneuploidy giving rise to aneuploid embryos, we evaluate the role of the variants in Meiosis I. However, it is possible that the chromosome segregation mistake arose during Meiosis II or in mitosis in the preimplantation embryo. Their implications in human female meiosis and aneuploidy risk remain to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The data provide evidence that gene variants exist in reproductively younger or advanced aged women that are predictive of the risk of producing aneuploid concepti in humans. Furthermore, a single amino acid in the N-terminus of AURKB is a gain-of-function mutant that could be protective of euploidy. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a Research Grant from the American Society of Reproductive Medicine and support from the Charles and Johanna Busch Memorial Fund at Rutgers, the State University of NJ to K.S. and the Foundation for Embryonic Competence, Inc to N.T. The authors declare no conflicts of interest.

Deficiency of the alkaline ceramidase ACER3 manifests in early childhood by progressive leukodystrophy.

BACKGROUND/AIMS: Leukodystrophies due to abnormal production of myelin cause extensive morbidity in early life; their genetic background is still largely unknown. We aimed at reaching a molecular diagnosis in Ashkenazi-Jewish patients who suffered from developmental regression at 6-13 months, leukodystrophy and peripheral neuropathy. METHODS: Exome analysis, determination of alkaline ceramidase activity catalysing the conversion of C18:1-ceramide to sphingosine and D-ribo-C12-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phytoceramide to NBD-C12-fatty acid using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and thin layer chromatography, respectively, and sphingolipid analysis in patients' blood by LC-MS/MS. RESULTS: The patients were homozygous for p.E33G in the ACER3, which encodes a C18:1-alkaline ceramidase and C20:1-alkaline ceramidase. The mutation abolished ACER3 catalytic activity in the patients' cells and failed to restore alkaline ceramidase activity in yeast mutant strain. The levels of ACER3 substrates, C18:1-ceramides and dihydroceramides and C20:1-ceramides and dihydroceramides and other long-chain ceramides and dihydroceramides were markedly increased in the patients' plasma, along with that of complex sphingolipids, including monohexosylceramides and lactosylceramides. CONCLUSIONS: Homozygosity for the p.E33G mutation in the ACER3 gene results in inactivation of ACER3, leading to the accumulation of various sphingolipids in blood and probably in brain, likely accounting for this new form of childhood leukodystrophy.

Leukoencephalopathy and early death associated with an Ashkenazi-Jewish founder mutation in the Hikeshi gene.

BACKGROUND: Leukodystrophies are genetic white matter disorders affecting the formation or maintenance of myelin. Among the recently discovered genetic defects associated with leukodystrophies, several genes converge on a common mechanism involving protein transcription/translation and ER stress response. METHODS: The genetic basis of a novel congenital leukodystrophy, associated with early onset spastic paraparesis, acquired microcephaly and optic atrophy was studied in six patients from three unrelated Ashkenazi-Jewish families. To this end we used homozygosity mapping, exome analysis, western blot (Hikeshi, HSF1-pS326 and b-actin) in patient fibroblasts, indirect immunofluorescence (HSP70 and HSF1) in patient fibroblasts undergoing heat shock stress, nuclear injection of plasmids expressing Hikeshi or EGFP in patient fibroblasts, in situ hybridization and Immunoblot analysis of Hikeshi in newborn and adult mouse brain. RESULTS: All the patients were homozygous for a missense mutation, p.Val54Leu, in C11ORF73 encoding HSP70 nuclear transporter protein, Hikeshi. The mutation segregated with the disease in the families and was carried by 1:200 Ashkenazi-Jewish individuals. The mutation was associated with undetectable level of Hikeshi in the patients' fibroblasts and with lack of nuclear HSP70 during heat shock stress, a phenomenon which was reversed upon the introduction of normal human Hikeshi to the patients cells. Hikeshi was found to be expressed in central white matter of mouse brain. CONCLUSIONS: These data underscore the importance of Hikeshi for HSP70 relocation into the nucleus. It is likely that in the absence of Hikeshi, HSP70 cannot attenuate the multiple heat shock induced nuclear phenotypes, leaving the cells unprotected during heat shock stress. We speculate that the sudden death of three of the six patients following a short febrile illness and the life-threatening myo-pericarditis in the fourth are the result of excess extra-nuclear HSP70 level which initiates cytokine release or provide target for natural killer cells. Alternatively, nuclear HSP70 might play an active role in stressed cells protection.

Mutations in SLC35A3 cause autism spectrum disorder, epilepsy and arthrogryposis.

BACKGROUND: The heritability of autism spectrum disorder is currently estimated at 55%. Identification of the molecular basis of patients with syndromic autism extends our understanding of the pathogenesis of autism in general. The objective of this study was to find the gene mutated in eight patients from a large kindred, who suffered from autism spectrum disorder, arthrogryposis and epilepsy. METHODS AND RESULTS: By linkage analysis and exome sequencing, we identified deleterious mutations in SLC35A3 in these patients. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter. In Golgi vesicles isolated from patient fibroblasts the transport of the respective nucleotide sugar was significantly reduced causing a massive decrease in the content of cell surface expressed highly branched N-glycans and a concomitant sharp increase of lower branched glycoforms. CONCLUSIONS: Spontaneous mutation in SLC35A3 has been discovered in cattle worldwide, recapitulating the human phenotype with arthrogryposis and additional skeletal defects known as Complex Vertebral Malformation syndrome. The skeletal anomalies in the mutant cattle and in our patients, and perhaps even the neurological symptoms are likely the consequence of the lack of high-branched N-glycans and the concomitant abundance of lower-branched glycoforms at the cell surface. This pattern has previously been associated with growth arrest and induction of differentiation. With this study, we add SLC35A3 to the gene list of autism spectrum disorders, and underscore the crucial importance of UDP-GlcNAc in the regulation of the N-glycan branching pathway in the Golgi apparatus.

Lack of association of KATNAL1 gene sequence variants and azoospermia in humans.

PURPOSE: A recent experiment indicated that a loss of function mutation in the murine Katnal1 gene resulted in male factor infertility due to premature exfoliation of spermatids. This study investigated the relevance of this gene to infertility in humans. METHODS: Multiple methods of genetic analysis were employed to investigate whether mutations in human KATNAL1 have a causative role in male infertility. This was a genetic association study, which included DNA samples from 105 men with non-obstructive azoospermia (NOA) and 242 anonymous sperm donor controls. 28 commercially available TaqMan SNP assays were used to haplotype samples from both groups and genetically tag regions of interest across the entire gene. AmpliSeq primers were then designed for identified regions so that targeted next-generation sequencing (NGS) could be used to identify causative variants. RESULTS: Four SNPs in the 3'UTR demonstrated a putative association with NOA. The AmpliSeq primers designed for the 3'UTR provided 83 % coverage of the 7,202 basepairs within the regions of interest. Variant sites were analyzed against genetic models to identify sequence polymorphisms which associated with NOA. No variants met standard criteria for significance when tested between the groups. CONCLUSIONS: This study suggests a lack of association of KATNAL1 gene sequence variants and azoospermia in humans.

Development of TaqMan allelic discrimination based genotyping of large DNA deletions.

The high prevalence of genetic diseases resulting from gross deletions has highlighted a need for a quick, simple, and reliable method of genotyping these mutations. Here, we developed a novel strategy for applying TaqMan allelic discrimination to accurately genotype 3 different large deletions in a high-throughput manner. Allelic discrimination has previously been used to genotype frame shift and point mutations, and small insertions or deletions six base pairs in length, but not large deletions. The assays designed here recognize a 2502 base pair deletion in the Nebulin (NEB) gene that results in Nemaline Myopathy, a 308,769 base pair deletion in the Gap Junction Protein, beta 6 (GJB6) gene that causes Hearing Loss, and a 6433 base pair deletion in the Mucolipin 1 (MCOLN1) gene responsible for causing Mucolipidosis IV Disease. This methodology may also be successfully applied to high throughput genotyping of other large deletions.

Prenatal Diagnosis of Cystic Fibrosis.

Cystic fibrosis (CF) is an inherited disease characterized by the accumulation of thick, sticky mucus which damages epithelia in organs such as the lungs, pancreas, liver, intestines, and other parts of the body. The most common symptoms are sinopulmonary disease and chronic gastrointestinal tract problems resulting from decreased mucociliary clearance and inflammation. CF is the most common life-limiting autosomal recessive disorder in people of northern European ancestry and it affects other populations with different prevalence. CF can be diagnosed by many methods including testing for blood immunoreactive trypsin, sweat chloride, transepithelial nasal potential difference, and molecular genetic testing.

Identification of a novel pathogenic OTOF variant causative of nonsyndromic hearing loss with high frequency in the Ashkenazi Jewish population.

Mutations in the OTOF gene have previously been shown to cause nonsyndromic prelingual deafness (DFNB9, OMIM 601071) as well as auditory neuropathy/dys-synchrony. In this study, the OTOF NM_194248.2 c.5332G>T, p.Val1778Phe variant was identified in a large Ashkenazi Jewish family as the causative variant in four siblings with hearing loss. Our analysis reveals a carrier frequency of the OTOF c.5332G>T, p.Val1778Phe variant of 1.27% in the Ashkenazi Jewish population, suggesting that this variant may be a significant contributor to nonsyndromic sensorineural hearing loss and should be considered for inclusion in targeted hearing loss panels for this population. Of note, the degree of hearing loss associated with this phenotype ranged from mild to moderately severe, with two of the four siblings not known to have hearing loss until they were genotyped and underwent pure tone audiometry and auditory brainstem response testing. The phenotypic variability along with the auditory neuropathy/dys-synchrony, which allows for the production of otoacoustic emissions, supports that nonsyndromic hearing loss caused by OTOF mutations may be much more common in the Ashkenazi Jewish population than currently appreciated due to a lack of diagnosis.

Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification.

OBJECTIVE: To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). DESIGN: Prospective and blinded. SETTING: Not applicable. PATIENT(S): Couples indicated for preimplantation genetic diagnosis (PGD). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. RESULT(S): The ADO frequency on a single cell from a fibroblast cell line was 1.64% (18/1,096). When two or more cells were tested, the ADO frequency dropped to 0.02% (1/4,426). The rate of amplification failure was 1.38% (55/4,000) overall, with 2.5% (20/800) for single cells and 1.09% (35/3,200) for samples that had two or more cells. Among 152 embryos tested in 17 cases by qPCR-based PGD and CCS, 100% were successfully given a diagnosis, with 0% ADO or amplification failure. Genotyping consistency with reference laboratory results was >99%. Another 304 embryos from 43 cases were included in the clinical application of qPCR-based PGD and CCS, for which 99.7% (303/304) of the embryos were given a definitive diagnosis, with only 0.3% (1/304) having an inconclusive result owing to recombination. In patients receiving a transfer with follow-up, the pregnancy rate was 82% (27/33). CONCLUSION(S): This study demonstrates that the use of qPCR for PGD testing delivers consistent and more reliable results than existing methods and that single gene disorder PGD can be run concurrently with CCS without the need for additional embryo biopsy or whole genome amplification.

Analytical Validation of a Personalized Medicine APOL1 Genotyping Assay for Nondiabetic Chronic Kidney Disease Risk Assessment.

The incidence of chronic kidney disease (CKD) varies by ancestry, with African Americans (AA) having a threefold to fourfold higher rate than whites. Notably, two APOL1 alleles, termed G1 [c.(1072A>G; 1200T>G)] and G2 (c.1212_1217del6), are strongly associated with higher rates of nondiabetic CKD and an increased risk for hypertensive end-stage renal disease. This has prompted the opportunity to implement APOL1 testing to identify at-risk patients and modify other risk factors to reduce the progression of CKD to end-stage renal disease. We developed an APOL1 genotyping assay using multiplex allele-specific primer extension, and validated using 58 positive and negative controls. Genotyping results were completely concordant with Sanger sequencing, and both triplicate interrun and intrarun genotyping results were completely concordant. Multiethnic APOL1 allele frequencies were also determined by genotyping 7059 AA, Hispanic, and Asian individuals from the New York City metropolitan area. The AA, Hispanic, and Asian APOL1 G1 and G2 allele frequencies were 0.22 and 0.13, 0.037 and 0.025, and 0.013 and 0.004, respectively. Notably, approximately 14% of the AA population carried two risk alleles and are at increased risk for CKD, compared with <1% of the Hispanic and Asian populations. This novel APOL1 genotyping assay is robust and highly accurate, and represents one of the first personalized medicine clinical genetic tests for disease risk prediction.

Carrier frequencies of eleven mutations in eight genes associated with primary ciliary dyskinesia in the Ashkenazi Jewish population.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal recessive disorder that results from functional and ultrastructural abnormalities of motile cilia. Patients with PCD have diverse clinical phenotypes that include chronic upper and lower respiratory tract infections, situs inversus, heterotaxy with or without congenital heart disease, and male infertility, among others. In this report, the carrier frequencies for eleven mutations in eight PCD-associated genes (DNAI1, DNAI2, DNAH5, DNAH11, CCDC114, CCDC40, CCDC65, and C21orf59) that had been found in individuals of Ashkenazi Jewish descent were investigated in order to advise on including them in existing clinical mutation panels for this population. Results showed relatively high carrier frequencies for the DNAH5 c.7502G>C mutation (0.58%), the DNAI2 c.1304G>A mutation (0.50%), and the C21orf59 c.735C>G mutation (0.48%), as well as lower frequencies for mutations in DNAI1, CCDC65, CCDC114, and DNAH11 (0.10-0.29%). These results suggest that several of these genes should be considered for inclusion in carrier screening panels in the Ashkenazi Jewish population.

High-throughput carrier screening using TaqMan allelic discrimination.

Members of the Ashkenazi Jewish community are at an increased risk for inheritance of numerous genetic diseases such that carrier screening is medically recommended. This paper describes the development and evaluation of 30 TaqMan allelic discrimination qPCR assays for 29 mutations on 2 different high-throughput platforms. Four of these mutations are in the GBA gene and are successfully examined using short amplicons due to the qualitative nature of TaqMan allelic discrimination. Two systems were tested for their reliability (call rate) and consistency with previous diagnoses (diagnostic accuracy) indicating a call rate of 99.04% and a diagnostic accuracy of 100% (+/-0.00%) from one platform, and a call rate of 94.66% and a diagnostic accuracy of 93.35% (+/-0.29%) from a second for 9,216 genotypes. Results for mutations tested at the expected carrier frequency indicated a call rate of 97.87% and a diagnostic accuracy of 99.96% (+/-0.05%). This study demonstrated the ability of a high throughput qPCR methodology to accurately and reliably genotype 29 mutations in parallel. The universally applicable nature of this technology provides an opportunity to increase the number of mutations that can be screened simultaneously, and reduce the cost and turnaround time for accommodating newly identified and clinically relevant mutations.

Evaluation of targeted next-generation sequencing-based preimplantation genetic diagnosis of monogenic disease.

OBJECTIVE: To investigate the applicability of next-generation sequencing (NGS) to preimplantation genetic diagnosis (PGD); to evaluate semiconductor-based NGS for genetic analysis of human embryos. DESIGN: Blinded. SETTING: Academic center for reproductive medicine. PATIENT(S): Six couples at risk of transmitting single-gene disorders to their offspring. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryonic genotype consistency of NGS with two independent conventional methods of PGD. RESULT(S): NGS provided 100% equivalent PGD diagnoses of compound point mutations and small deletions and insertions compared with both reference laboratory- and internally developed quantitative polymerase chain reaction (qPCR)-based analyses. Furthermore, NGS single-gene disorder screening could be performed in parallel with qPCR-based comprehensive chromosome screening. CONCLUSION(S): NGS can provide blastocyst PGD results with a high level of consistency with established methodologies. This study and its design could serve as a model for further development of this important and emerging technology.

High-throughput real-time PCR-based genotyping without DNA purification.

BACKGROUND: While improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform. FINDINGS: The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%. CONCLUSION: This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification.