Purpose The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for the translocation of misfolded proteins across the ER membrane into the cytosol for subsequent degradation by the proteasome. In order to understand the spectrum of clinical and molecular findings in a complex neurological syndrome, we studied a series of eight patients with inherited deficiency of N-glycanase 1 (NGLY1), a novel disorder of cytosolic ERAD dysfunction. Methods Whole-genome, whole-exome or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data. Results All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypo- or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele. Conclusions NGLY1 deficiency is a novel autosomal recessive disorder of the ERAD pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a more broad range of mutations are detected.

The Roche PGMY primer-based research prototype line blot assay (PGMY-LB) is a convenient tool in epidemiological studies for the detection and typing of human papillomavirus (HPV) DNA. This assay has been optimized and is being commercialized as the Linear Array HPV genotyping test (LA-HPV). We assessed the agreement between LA-HPV and PGMY-LB for detection and typing of 37 HPV genotypes in 528 anogenital samples ( Infection by human papillomavirus (HPV) causes squamous intraepithelial lesions and invasive cancer of the uterine cervix and anus (3). HPV testing relies on the detection and analysis of viral DNA. Epidemiological studies and vaccine clinical trials require reliable and reproducible identification and genotyping of genital HPV infections. Since only a fraction of the 40 HPV genotypes infecting the anogenital tract are associated with malignant lesions, the detection method has to identify types individually. Specific genotyping also provides information on mixed HPV infections (26). Type-specific PCR assays are impractical for epidemiological studies because of the multiplicity of relevant genotypes infecting the anogenital tract. Consensus PCR assays that target conserved regions of the HPV genome have been devised to amplify all relevant genital types in one reaction, with analysis of amplicons by direct sequencing, restriction fragment length polymorphism analysis, or type-specific hybridization.The most common PCR methods use the consensus primer set MY09/MY11/HMB01 (20, 25), GP5ϩ/GP6ϩ (9, 21), PGMY09/ PGMY11 (13,34), or SPF10 (30,34). Convenient assays for detection and typing of HPV have been developed for all of these primer sets. HPV amplicons generated by PGMY or MY primers can easily be detected and typed by a nonisotopic

C ervical cancer is caused by persistent infection with high-risk human papillomavirus (hrHPV). The incidence of invasive cervical cancer has been significantly reduced over the past 50 years by the widespread implementation of cytology-based screening using the Papanicolaou (Pap) test (1). However, because of the limited sensitivity of a single Pap test, cervical cancer and its precursors (cervical intraepithelial neoplasia grades 2 and 3 [CIN2 and CIN3, respectively]) remain significant public health issues. The recent development of assays that allow the detection of hrHPV DNA in cervical specimens has shown them to be more sensitive than the Pap test for the detection of CIN2 and CIN3 in a single screening round (2-4). To be clinically relevant, HPV assays should be based on clinical rather than on analytic sensitivity, since only a minority of HPV-positive women progress to highgrade cervical disease. Moreover, the clinical utility of an HPV test would be enhanced if it could also identify hrHPV-positive women who are at the highest risk of having or developing highgrade disease, because doing so has the potential to improve both sensitivity and specificity. Given the recent evidence for the significantly greater oncogenic risks of HPV16 and HPV18 relative to the other hrHPV types (5-8), an HPV test capable of specifically identifying these 2 genotypes might be expected to provide additional clinical value. Moreover, all HPV tests must demonstrate analytic sensitivity, accuracy, good reproducibility, and inclusivity and exclusivity.The cobas HPV test is a fully automated real-time PCR DNA amplification test that received approval by the Food and Drug Administration in April 2011 and was developed to maximize clinical utility. The cutoff for a positive result was determined based on the ability of the assay to detect histologically confirmed high-grade cervical disease, defined as cervical intraepithelial neoplasia (CIN), grade 2 or worse (ՆCIN2), rather than on the detection of a minimum number of viral copies. The unique test design also allowed simultaneous reporting of a pooled hrHPV result in addition to individual results for HPV16 and HPV18, the 2 most-oncogenic genotypes (5-8). Here, we report details on the development and validation of the cobas HPV test with emphasis on how its design and performance contribute to the establishment of clinical utility. MATERIALS AND METHODSAssay design and conditions. The cobas HPV test is a highly automated assay for the detection of hrHPV DNA in liquid-based cytology (LBC) specimens using real-time PCR technology with a set of 16 PCR primers (8 forward and 8 reverse) that amplify a ϳ200-bp fragment of the L1 gene from all 14 hrHPV genotypes. TaqMan probes labeled with 3 spectrally unique fluorescent dyes allow for the simultaneous detection of 14 hrHPV types from 3 separate channels with real-time PCR technology. Twelve hrHPV types (HPV31, are detected as a pool in channel 1, and the hrHPV16 and HPV18 genotypes are simultaneously detected individually in channel...

Our analytical framework allows estimation of HGAIN cases attributable to individual HPV genotypes in the context of multiple concurrent HPV infections, which are very common among HIV-infected MSM. Our results suggest that licensed and investigational HPV prophylactic vaccines have the potential to prevent a substantial proportion of HGAIN cases in this population.

Background Although human leukocyte antigen (HLA) DQ and DR loci appear to confer the strongest genetic risk for type 1 diabetes, more detailed information is required for other loci within the HLA region to understand causality and stratify additional risk factors. The Type 1 Diabetes Genetics Consortium (T1DGC) study design included high-resolution genotyping of HLA-A, B, C, DRB1, DQ, and DP loci in all affected sibling pair and trio families, and cases and controls, recruited from four networks worldwide, for analysis with clinical phenotypes and immunological markers.Purpose In this article, we present the operational strategy of training, classification, reporting, and quality control of HLA genotyping in four laboratories on three continents over nearly 5 years.Methods Methods to standardize HLA genotyping at eight loci included: central training and initial certification testing; the use of uniform reagents, protocols, instrumentation, and software versions; an automated data transfer; and the use of standardized nomenclature and allele databases. We implemented a rigorous and consistent quality control process, reinforced by repeated workshops, yearly meetings, and telephone conferences.Results A total of 15,246 samples have been HLA genotyped at eight loci to four-digit resolution; an additional 6797 samples have been HLA genotyped at two loci. The genotyping repeat rate decreased significantly over time, with an estimated unresolved Mendelian inconsistency rate of 0.21%. Annual quality control exercises tested 2192 genotypes (4384 alleles) and achieved 99.82% intra-laboratory and 99.68% inter-laboratory concordances.Limitations The chosen genotyping platform was unable to distinguish many allele combinations, which would require further multiple stepwise testing to resolve. For these combinations, a standard allele assignment was agreed upon, allowing further analysis if required.Conclusions High-resolution HLA genotyping can be performed in multiple laboratories using standard equipment, reagents, protocols, software, and communication to produce consistent and reproducible data with minimal systematic error. Many of the strategies used in this study are generally applicable to other large multi-center studies.