The Alu-rich genomic architecture of SPAST predisposes to diverse and functionally distinct disease-associated CNV alleles.

Intragenic copy-number variants (CNVs) contribute to the allelic spectrum of both Mendelian and complex disorders. Although pathogenic deletions and duplications in SPAST (mutations in which cause autosomal-dominant spastic paraplegia 4 [SPG4]) have been described, their origins and molecular consequences remain obscure. We mapped breakpoint junctions of 54 SPAST CNVs at nucleotide resolution. Diverse combinations of exons are deleted or duplicated, highlighting the importance of particular exons for spastin function. Of the 54 CNVs, 38 (70%) appear to be mediated by an Alu-based mechanism, suggesting that the Alu-rich genomic architecture of SPAST renders this locus susceptible to various genome rearrangements. Analysis of breakpoint Alus further informs a model of Alu-mediated CNV formation characterized by small CNV size and potential involvement of mechanisms other than homologous recombination. Twelve deletions (22%) overlap part of SPAST and a portion of a nearby, directly oriented gene, predicting novel chimeric genes in these subjects' genomes. cDNA from a subject with a SPAST final exon deletion contained multiple SPAST:SLC30A6 fusion transcripts, indicating that SPAST CNVs can have transcriptional effects beyond the gene itself. SLC30A6 has been implicated in Alzheimer disease, so these fusion gene data could explain a report of spastic paraplegia and dementia cosegregating in a family with deletion of the final exon of SPAST. Our findings provide evidence that the Alu genomic architecture of SPAST predisposes to diverse CNV alleles with distinct transcriptional--and possibly phenotypic--consequences. Moreover, we provide further mechanistic insights into Alu-mediated copy-number change that are extendable to other loci.

A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders.

We developed a rules-based scoring system to classify DNA variants into five categories including pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Over 16,500 pathogenicity assessments on 11,894 variants from 338 genes were analyzed for pathogenicity based on prediction tools, population frequency, co-occurrence, segregation, and functional studies collected from internal and external sources. Scores were calculated by trained scientists using a quantitative framework that assigned differential weighting to these five types of data. We performed descriptive and comparative statistics on the dataset and tested interobserver concordance among the trained scientists. Private variants defined as variants found within single families (n = 5,182), were either VUS (80.5%; n = 4,169) or likely pathogenic (19.5%; n = 1,013). The remaining variants (n = 6,712) were VUS (38.4%; n = 2,577) or likely benign/benign (34.7%; n = 2,327) or likely pathogenic/pathogenic (26.9%, n = 1,808). Exact agreement between the trained scientists on the final variant score was 98.5% [95% confidence interval (CI) (98.0, 98.9)] with an interobserver consistency of 97% [95% CI (91.5, 99.4)]. Variant scores were stable and showed increasing odds of being in agreement with new data when re-evaluated periodically. This carefully curated, standardized variant pathogenicity scoring system provides reliable pathogenicity scores for DNA variants encountered in a clinical laboratory setting.

Characteristics of maturity onset diabetes of the young in a large diabetes center.

Maturity onset diabetes of the young (MODY) is a monogenic form of diabetes caused by a mutation in a single gene, often not requiring insulin. The aim of this study was to estimate the frequency and clinical characteristics of MODY at the Barbara Davis Center. A total of 97 subjects with diabetes onset before age 25, a random C-peptide ≥0.1 ng/mL, and negative for all diabetes autoantibodies (GADA, IA-2, ZnT8, and IAA) were enrolled, after excluding 21 subjects with secondary diabetes or refusal to participate. Genetic testing for MODY 1-5 was performed through Athena Diagnostics, and all variants of unknown significance were further analyzed at Exeter, UK. A total of 22 subjects [20 (21%) when excluding two siblings] were found to have a mutation in hepatocyte nuclear factor 4A (n = 4), glucokinase (n = 8), or hepatocyte nuclear factor 1A (n = 10). Of these 22 subjects, 13 had mutations known to be pathogenic and 9 (41%) had novel mutations, predicted to be pathogenic. Only 1 of the 22 subjects had been given the appropriate MODY diagnosis prior to testing. Compared with MODY-negative subjects, the MODY-positive subjects had lower hemoglobin A1c level and no diabetic ketoacidosis at onset; however, these characteristics are not specific for MODY. In summary, this study found a high frequency of MODY mutations with the majority of subjects clinically misdiagnosed. Clinicians should have a high index of suspicion for MODY in youth with antibody-negative diabetes.

Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer.

PALB2 was recently identified as a nuclear binding partner of BRCA2. Biallelic BRCA2 mutations cause Fanconi anemia subtype FA-D1 and predispose to childhood malignancies. We identified pathogenic mutations in PALB2 (also known as FANCN) in seven families affected with Fanconi anemia and cancer in early childhood, demonstrating that biallelic PALB2 mutations cause a new subtype of Fanconi anemia, FA-N, and, similar to biallelic BRCA2 mutations, confer a high risk of childhood cancer.

The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia.

Seven Fanconi anemia-associated proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) form a nuclear Fanconi anemia core complex that activates the monoubiquitination of FANCD2, targeting FANCD2 to BRCA1-containing nuclear foci. Cells from individuals with Fanconi anemia of complementation groups D1 and J (FA-D1 and FA-J) have normal FANCD2 ubiquitination. Using genetic mapping, mutation identification and western-blot data, we identify the defective protein in FA-J cells as BRIP1 (also called BACH1), a DNA helicase that is a binding partner of the breast cancer tumor suppressor BRCA1.

Mechanisms for nonrecurrent genomic rearrangements associated with CMT1A or HNPP: rare CNVs as a cause for missing heritability.

Genomic rearrangements involving the peripheral myelin protein gene (PMP22) in human chromosome 17p12 are associated with neuropathy: duplications cause Charcot-Marie-Tooth disease type 1A (CMT1A), whereas deletions lead to hereditary neuropathy with liability to pressure palsies (HNPP). Our previous studies showed that >99% of these rearrangements are recurrent and mediated by nonallelic homologous recombination (NAHR). Rare copy number variations (CNVs) generated by nonrecurrent rearrangements also exist in 17p12, but their underlying mechanisms are not well understood. We investigated 21 subjects with rare CNVs associated with CMT1A or HNPP by oligonucleotide-based comparative genomic hybridization microarrays and breakpoint sequence analyses, and we identified 17 unique CNVs, including two genomic deletions, ten genomic duplications, two complex rearrangements, and three small exonic deletions. Each of these CNVs includes either the entire PMP22 gene, or exon(s) only, or ultraconserved potential regulatory sequences upstream of PMP22, further supporting the contention that PMP22 is the critical gene mediating the neuropathy phenotypes associated with 17p12 rearrangements. Breakpoint sequence analysis reveals that, different from the predominant NAHR mechanism in recurrent rearrangement, various molecular mechanisms, including nonhomologous end joining, Alu-Alu-mediated recombination, and replication-based mechanisms (e.g., FoSTeS and/or MMBIR), can generate nonrecurrent 17p12 rearrangements associated with neuropathy. We document a multitude of ways in which gene function can be altered by CNVs. Given the characteristics, including small size, structural complexity, and location outside of coding regions, of selected rare CNVs, their identification remains a challenge for genome analysis. Rare CNVs may potentially represent an important portion of "missing heritability" for human diseases.

Functional assessment of TSC1 missense variants identified in individuals with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Previously, we demonstrated that pathogenic amino acid substitutions in the N-terminal domain of TSC1 (amino acids 50-224) are destabilizing. Here we investigate an additional 21 unclassified TSC1 variants. Our functional assessment identified four substitutions (p.L61R, p.G132D, p.F158S, and p.R204P) between amino acids 50 and 224 that reduced TSC1 stability and prevented the TSC1-TSC2-dependent inhibition of TORC1. In four cases (20%), our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our new data confirm our previous finding that the N-terminal region of TSC1 is essential for TSC1 function.

Technical standards and guidelines for spinal muscular atrophy testing.

Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron (SMN1) gene, affecting approximately 1 in 10,000 live births. The disease is characterized by progressive symmetrical muscle weakness resulting from the degeneration and loss of anterior horn cells in the spinal cord and brainstem nuclei. The disease is classified on the basis of age of onset and clinical course. Two almost identical SMN genes are present on 5q13: the SMN1 gene, which is the spinal muscular atrophy-determining gene, and the SMN2 gene. The homozygous absence of the SMN1 exon 7 has been observed in the majority of patients and is being used as a reliable and sensitive spinal muscular atrophy diagnostic test. Although SMN2 produces less full-length transcript than SMN1, the number of SMN2 copies has been shown to modulate the clinical phenotype. Carrier detection relies on the accurate determination of the SMN1 gene copies. This document follows the outline format of the general Standards and Guidelines for Clinical Laboratories. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and methods of analysis.

Alu-specific microhomology-mediated deletion of the final exon of SPAST in three unrelated subjects with hereditary spastic paraplegia.

PURPOSE: Autosomal dominant spastic paraplegia, type 4 (SPG4), a debilitating disorder of progressive spasticity and weakness of the lower limbs, results from heterozygous mutations in the SPAST gene. The full spectrum of SPAST mutations causing SPG4 and their mechanisms of formation remain to be determined. METHODS: We used multiplex ligation-dependent probe amplification, locus-specific array comparative genomic hybridization, and breakpoint DNA sequencing to identify and describe genomic rearrangements in three patients with a clinical presentation of hereditary spastic paraplegia. RESULTS: We describe three SPG4 patients with intragenic rearrangements in SPAST; all specifically delete the final exon, exon 17. Breakpoint sequence analyses provide evidence for Alu-specific microhomology-mediated deletion as the mechanism of exon loss; one complex rearrangement apparently occurred by multiple Alu-facilitated template switches. CONCLUSION: We hypothesize that the high concentration of Alu family members in the introns and flanking sequence of SPAST may predispose to intragenic rearrangements. Thus, Alu-specific microhomology-mediated intragenic rearrangements in SPAST may be a common cause of SPG4. Furthermore, we propose that genomic deletions encompassing the final exon of SPAST may affect expression of SLC30A6, the most proximal downstream locus and a gene that has been implicated in the pathogenesis of Alzheimer disease, potentially explaining recent reports of dementia in selected SPG4 patients.

The Alzheimer's Association external quality control program for cerebrospinal fluid biomarkers.

BACKGROUND: The cerebrospinal fluid (CSF) biomarkers amyloid ß (Aß)-42, total-tau (T-tau), and phosphorylated-tau (P-tau) demonstrate good diagnostic accuracy for Alzheimer's disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimer's Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch-to-batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program. METHODS: The program is open for laboratories using commercially available kits for Aß, T-tau, or P-tau. CSF samples (aliquots of pooled CSF) are sent for analysis several times a year from the Clinical Neurochemistry Laboratory at the Mölndal campus of the University of Gothenburg, Sweden. Each round consists of three quality control samples. RESULTS: Forty laboratories participated. Twenty-six used INNOTEST enzyme-linked immunosorbent assay kits, 14 used Luminex xMAP with the INNO-BIA AlzBio3 kit (both measure Aß-(1-42), P-tau(181P), and T-tau), and 5 used Meso Scale Discovery with the Aß triplex (AßN-42, AßN-40, and AßN-38) or T-tau kits. The total coefficients of variation between the laboratories were 13% to 36%. Five laboratories analyzed the samples six times on different occasions. Within-laboratory precisions differed considerably between biomarkers within individual laboratories. CONCLUSIONS: Measurements of CSF AD biomarkers show large between-laboratory variability, likely caused by factors related to analytical procedures and the analytical kits. Standardization of laboratory procedures and efforts by kit vendors to increase kit performance might lower variability, and will likely increase the usefulness of CSF AD biomarkers.

Limbic encephalitis in a child with ovarian teratoma and influenza B. Case report and critical review of the history of autoimmune anti-N-methyl-d-aspartate receptor encephalitis.

We report the appearance of clinical symptoms and signs of N-methyl-d-Aspartate (NMDA) receptor encephalitis in a patient presenting just days after contraction of influenza B. The offending mature ovarian teratoma was identified and removed on the 10th day after the appearance of symptoms, with subsequent nearly complete resolution of symptoms over the subsequent 6 months. We provide a focused literature review of the clinical and pathophysiologic literature of anti-NMDA receptor encephalitis pertaining to influenza B virus and the pediatric population. Taken together, this study contributes to the pathophysiological understanding of anti-NMDA receptor encephalitis and aids clinicians in its early recognition and management.

GJB1/Connexin 32 whole gene deletions in patients with X-linked Charcot-Marie-Tooth disease.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is the second most common form of this genetically heterogeneous inherited peripheral neuropathy. CMT1X is caused by mutations in the GJB1 gene. Most of the mutations causative for CMT1X are missense mutations. In addition, a few disease causative nonsense mutations and frameshift deletions that lead to truncated forms of the protein have also been reported to be associated with CMT1X. Previously, there have been reports of patients with deletions of the coding sequence of GJB1; however, the size and breakpoints of these deletions were not assessed. Here, we report five patients with deletions that range in size from 12.2 to 48.3 kb and that completely eliminate the entire coding sequence of the GJB1 gene, resulting in a null allele for this locus. Analyses of the breakpoints of these deletions showed that they are nonrecurrent and that they can be generated by different mechanisms. In addition to PMP22, GJB1 is the second CMT gene for which both point mutations and genomic rearrangements can cause a neuropathy phenotype, stressing the importance of CMT as a genomic disorder.

High-throughput mutational analysis of TOR1A in primary dystonia.

BACKGROUND: Although the c.904_906delGAG mutation in Exon 5 of TOR1A typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A) has been associated with early-onset generalized dystonia and some DeltaGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify TOR1A Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dystonia. METHODS: High resolution melting (HRM) was used to examine the entire TOR1A Exon 5 coding sequence in 1014 subjects with primary dystonia (422 spasmodic dysphonia, 285 cervical dystonia, 67 blepharospasm, 41 writer's cramp, 16 oromandibular dystonia, 38 other primary focal dystonia, 112 segmental dystonia, 16 multifocal dystonia, and 17 generalized dystonia) and 250 controls (150 neurologically normal and 100 with other movement disorders). Diagnostic sensitivity and specificity were evaluated in an additional 8 subjects with known DeltaGAG DYT1 dystonia and 88 subjects with DeltaGAG-negative dystonia. RESULTS: HRM of TOR1A Exon 5 showed high (100%) diagnostic sensitivity and specificity. HRM was rapid and economical. HRM reliably differentiated the TOR1A DeltaGAG and c.863G>A mutations. Melting curves were normal in 250/250 controls and 1012/1014 subjects with primary dystonia. The two subjects with shifted melting curves were found to harbor the classic DeltaGAG deletion: 1) a non-Jewish Caucasian female with childhood-onset multifocal dystonia and 2) an Ashkenazi Jewish female with adolescent-onset spasmodic dysphonia. CONCLUSION: First, HRM is an inexpensive, diagnostically sensitive and specific, high-throughput method for mutation discovery. Second, Exon 5 mutations in TOR1A are rarely associated with non-generalized primary dystonia.

The spectrum of SCN1A-related infantile epileptic encephalopathies.

The relationship between severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome) and the related syndrome SMEI-borderland (SMEB) with mutations in the sodium channel alpha 1 subunit gene SCN1A is well established. To explore the phenotypic variability associated with SCN1A mutations, 188 patients with a range of epileptic encephalopathies were examined for SCN1A sequence variations by denaturing high performance liquid chromatography and sequencing. All patients had seizure onset within the first 2 years of life. A higher proportion of mutations were identified in patients with SMEI (52/66; 79%) compared to patients with SMEB (25/36; 69%). By studying a broader spectrum of infantile epileptic encephalopathies, we identified mutations in other syndromes including cryptogenic generalized epilepsy (24%) and cryptogenic focal epilepsy (22%). Within the latter group, a distinctive subgroup designated as severe infantile multifocal epilepsy had SCN1A mutations in three of five cases. This phenotype is characterized by early onset multifocal seizures and later cognitive decline. Knowledge of an expanded spectrum of epileptic encephalopathies associated with SCN1A mutations allows earlier diagnostic confirmation for children with these devastating disorders.

Can Biomarkers Help Target Maturity-Onset Diabetes of the Young Genetic Testing in Antibody-Negative Diabetes?

BACKGROUND: Maturity-onset diabetes of the young (MODY) is an antibody-negative, autosomal dominant form of diabetes. With the increasing prevalence of diabetes and the expense of MODY testing, markers to identify those who need further genetic testing would be beneficial. We investigated whether HLA genotypes, random C-peptide, and/or high-sensitivity C-reactive protein (hsCRP) levels could be helpful biomarkers for identifying MODY in antibody-negative diabetes. METHODS: Subjects (N = 97) with diabetes onset ≤age 25, measurable C-peptide (≥0.1 ng/mL), and negative for all four diabetes autoantibodies were enrolled at a large academic center and tested for MODY 1-5 through Athena Diagnostics. A total of 22 subjects had a positive or very likely pathogenic mutation for MODY. RESULTS: Random C-peptide levels were significantly different between MODY-positive and MODY-negative subjects (0.16 nmol/L vs. 0.02 nmol/L; P = 0.02). After adjusting for age and diabetes duration, hsCRP levels were significantly lower in MODY-positive subjects (0.37 mg/L vs. 0.87 mg/L; P = 0.02). Random C-peptide level ≥0.15 nmol/L obtained at ≥6 months after diagnosis had 83% sensitivity for diagnosis of MODY with a negative predictive value of 96%. Receiver operating characteristic curves showed that area under the curve for random C-peptide (0.75) was significantly better than hsCRP (0.54), high-risk HLA DR3/4-DQB1*0302 (0.59), and high-risk HLA/random C-peptide combined (0.54; P = 0.03). CONCLUSIONS: Random C-peptide obtained at ≥6 months after diagnosis can be a useful biomarker to identify antibody-negative individuals who need further genetic testing for MODY, whereas hsCRP and HLA do not appear to improve this antibody/C-peptide-based approach.

Aprataxin (APTX) gene mutations resembling multiple system atrophy.

Mutations of the aprataxin (APTX) gene cause early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH), also called ataxia with oculomotor apraxia type 1. Recent studies showed clinical heterogeneity in patients with EAOH. We describe 2 patients whose clinical features resembled those of multiple system atrophy of the cerebellar subtype (MSA-C) but without ocular motor apraxia and hypoalbuminemia. Each had a different nucleotide transition in the APTX gene (725G-->A and 457A-->G). These variants on the APTX gene exhibit phenotypic variability.

Anti-MuSK myasthenia gravis presenting with purely ocular findings.

BACKGROUND: Antibodies to a muscle-specific receptor tyrosine kinase (MuSK) have been found in approximately 40% of patients with generalized myasthenia gravis who are seronegative for the antiacetylcholine receptor antibody. Many of the patients with anti-MuSK antibodies have prominent oculobulbar symptoms or weakness of the neck and respiratory muscles, but patients with ocular myasthenia have not been described. OBJECTIVE: To report a case of ocular myasthenia due to anti-MuSK antibodies. PATIENT: A young woman with ocular myasthenia and antibodies to MuSK. RESULTS: Anti-MuSK antibody was detected by radioimmunoassay using highly purified MuSK recombinant antigen. CONCLUSION: Ocular myasthenia gravis is a presentation of the anti-MuSK antibody syndrome.