Development and Validation of a Next-Generation Sequencing Panel for Syndromic and Nonsyndromic Hearing Loss.

BACKGROUND: Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. METHODS: We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. RESULTS: We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1-22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. CONCLUSIONS: We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

The Power of Proficiency Testing: Unraveling Single-Nucleotide Polymorphism Interference, With Potential Impact on Clinical Testing of Spinocerebellar Ataxia Type 3.

CONTEXT.­: The College of American Pathologists proficiency testing program has been instrumental in identifying problems in clinical testing. OBJECTIVE.­: To describe how this program was used to identify a single-nucleotide polymorphism that affects clinical testing for spinocerebellar ataxia type 3. DESIGN.­: A proficiency testing sample with discordant results for spinocerebellar ataxia type 3 analysis was further evaluated by targeted Sanger sequencing and genotype polymerase chain reaction using multiple DNA polymerases. RESULTS.­: Of 28 laboratories responding in the spinocerebellar ataxia type 3 Proficiency Survey, 18 reported an incorrect homozygous result and 10 reported the expected heterozygous result. A heterozygous single-nucleotide polymorphism complementary to the 3' end of a published forward primer was identified in the proficiency testing sample, which may have led to allele dropout. However, this primer was used by only 3 of 18 laboratories (16%) reporting a homozygous result. A new forward primer of identical sequence, except for the 3' end being complementary to the single-nucleotide polymorphism, showed the expected heterozygous pattern. The possibility of DNA polymerase 3'-5' exonuclease activity contributing to allele dropout was investigated by testing 9 additional polymerases with and without exonuclease activity. No clear pattern emerged, but enzymes with and without 3'-5' exonuclease activity yielded both homozygous and expected heterozygous results with the published forward primer. CONCLUSIONS.­: Proactive systematic primer sequence checking is recommended because single-nucleotide polymorphism interference may result in allele dropout and impact clinical testing. Allele dropout is also influenced by other factors, including DNA polymerase exonuclease activity.

C9orf72 Repeat Expansion Frequency among Patients with Huntington Disease Genetic Testing.

BACKGROUND: European studies identified the C9orf72 repeat expansion as the most frequent genetic alteration in patients with Huntington disease (HD)-like phenotypes but negative HD genetic testing. OBJECTIVE: To investigate C9orf72 repeat expansion frequency in individuals tested for HD in a North American tertiary referral laboratory. METHODS: Three hundred and seventy-three cases (115 positive and 258 negative for HD) were evaluated by genotyping PCR, with follow-up Southern blot and 5' repeat methylation status assessment by combined repeat-primed and methylation-specific PCR in a subset. RESULTS: Three cases (all HD-negative) tested positive: 2 had > 2,000 repeats and were methylated, 1 had 80-100 repeats and was unmethylated. Two cases (1 HD-positive and 1 HD-negative) had intermediate alleles (20-29 repeats) and were unmethylated. The remaining 368 cases were negative (< 20 repeats). C9orf72 repeat expansion was absent in patients with HD and was identified in a small subset (1.2%) of patients with negative HD genetic testing. CONCLUSION: These findings suggest that C9orf72 repeat expansion does not coexist with HTT repeat expansion and that C9orf72 repeat expansion testing is unnecessary for patients with HD. In addition, C9orf72 evaluation may be considered for individuals negative for HD genetic testing. Similar to in previous studies, methylation of C9orf72 repeat expansion was limited to large expansions.

Target-enrichment sequencing and copy number evaluation in inherited polyneuropathy.

OBJECTIVE: To assess the efficiency of target-enrichment next-generation sequencing (NGS) with copy number assessment in inherited neuropathy diagnosis. METHODS: A 197 polyneuropathy gene panel was designed to assess for mutations in 93 patients with inherited or idiopathic neuropathy without known genetic cause. We applied our novel copy number variation algorithm on NGS data, and validated the identified copy number mutations using CytoScan (Affymetrix). Cost and efficacy of this targeted NGS approach was compared to earlier evaluations. RESULTS: Average coverage depth was ∼760× (median = 600, 99.4% > 100×). Among 93 patients, 18 mutations were identified in 17 cases (18%), including 3 copy number mutations: 2 PMP22 duplications and 1 MPZ duplication. The 2 patients with PMP22 duplication presented with bulbar and respiratory involvement and had absent extremity nerve conductions, leading to axonal diagnosis. Average onset age of these 17 patients was 25 years (2-61 years), vs 45 years for those without genetic discovery. Among those with onset age less than 40 years, the diagnostic yield of targeted NGS approach is high (27%) and cost savings is significant (∼20%). However, the cost savings for patients with late onset age and without family history is not demonstrated. CONCLUSIONS: Incorporating copy number analysis in target-enrichment NGS approach improved the efficiency of mutation discovery for chronic, inherited, progressive length-dependent polyneuropathy diagnosis. The new technology is facilitating a simplified genetic diagnostic algorithm utilizing targeted NGS, clinical phenotypes, age at onset, and family history to improve diagnosis efficiency. Our findings prompt a need for updating the current practice parameters and payer guidelines.

Does parent of origin matter? Methylation studies should be performed on patients with multiple copies of the Prader-Willi/Angelman syndrome critical region.

Deletion of 15q11.2-q13 results in either Prader-Willi syndrome (PWS) or Angelman syndrome (AS) depending on the parent of origin. Duplication of the PWS/AS critical region (PWASCR) has also been reported in association with developmental delay and autism, and it has been shown that they also show a parent-of-origin effect. It is generally accepted that maternal duplications are pathogenic. However, there is conflicting evidence as to the pathogenicity of paternal duplications. We have identified 35 patients with gain of the PWASCR using array comparative genomic hybridization. Methylation testing was performed to determine parent of origin of the extra copies. Of the 35 cases, 22 had a supernumerary marker chromosome 15 (SMC15), 12 had a tandem duplication, and 1 had a tandem triplication. Only one patient had a paternal duplication; this patient does not have features typical of patients with maternal duplication of the PWASCR. Three of the mothers had a tandem duplication (two were paternal and one was maternal origin). While one of the two mothers with paternal duplication was noted not to have autism, the other was noted to have learning disability and depression. Based on our data, we conclude that SMC15 are almost exclusively maternal in origin and result in an abnormal phenotype. Tandem duplications/triplications are generally of maternal origin when ascertained on the basis of abnormal phenotype; however, tandem duplications of paternal origin have also been identified. Therefore, we suggest that methylation testing be performed for cases of tandem duplications/triplications since the pathogenicity of paternal gains is uncertain.

Development and clinical implementation of a combination deletion PCR and multiplex ligation-dependent probe amplification assay for detecting deletions involving the human α-globin gene cluster.

The α-thalassemias are a group of hereditary disorders caused by reduced synthesis of the α-chain of hemoglobin. We have developed and tested an α-thalassemia assay that uses both multiplex ligation-dependent probe amplification (MLPA) with Luminex-based detection and deletion PCR technologies. The MLPA assay consisted of 20 probes, 15 of which hybridized to the α-globin gene cluster and 5 that served as control probes. A PCR assay was developed to confirm the presence of heterozygous/homozygous 3.7-kb and 4.2-kb deletions. MLPA and PCR results were compared to Southern blot (SB) results from 758 and 133 specimens, respectively. Lastly, MLPA and PCR results were reviewed and summarized from 5386 clinically tested specimens. SB and MLPA results were concordant in 678/687 (99%) specimens. PCR detected all deletions detected by SB with no false positives. No deletions or duplications were identified in 2630 (49%) clinically tested specimens. Extra α-globin copies were identified in 76 patients. A deletion of one or two α-globin genes was identified in 1251 (23%) and 1349 (25%) specimens, respectively, including 15 different genotypes. A deletion of three (hemoglobin H) and four α-globin genes (Hb Bart's) was observed in 65 or 3 specimens, respectively. Six patients had a deletion within the α-globin regulatory region MCS-R2. Thus, MLPA plus deletion PCR identify multiple α-globin gene deletions/duplications in patients being tested for α-thalassemia.

Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome.

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.

Comprehensive mutation screening in 55 probands with type 1 primary hyperoxaluria shows feasibility of a gene-based diagnosis.

Mutations in AGXT, a locus mapped to 2q37.3, cause deficiency of liver-specific alanine:glyoxylate aminotransferase (AGT), the metabolic error in type 1 primary hyperoxaluria (PH1). Genetic analysis of 55 unrelated probands with PH1 from the Mayo Clinic Hyperoxaluria Center, to date the largest with availability of complete sequencing across the entire AGXT coding region and documented hepatic AGT deficiency, suggests that a molecular diagnosis (identification of two disease alleles) is feasible in 96% of patients. Unique to this PH1 population was the higher frequency of G170R, the most common AGXT mutation, accounting for 37% of alleles, and detection of a new 3' end deletion (Ex 11_3'UTR del). A described frameshift mutation (c.33_34insC) occurred with the next highest frequency (11%), followed by F152I and G156R (frequencies of 6.3 and 4.5%, respectively), both surpassing the frequency (2.7%) of I244T, the previously reported third most common pathogenic change. These sequencing data indicate that AGXT is even more variable than formerly believed, with 28 new variants (21 mutations and seven polymorphisms) detected, with highest frequencies on exons 1, 4, and 7. When limited to these three exons, molecular analysis sensitivity was 77%, compared with 98% for whole-gene sequencing. These are the first data in support of comprehensive AGXT analysis for the diagnosis of PH1, obviating a liver biopsy in most well-characterized patients. Also reported here is previously unavailable evidence for the pathogenic basis of all AGXT missense variants, including evolutionary conservation data in a multisequence alignment and use of a normal control population.

Identification of 58 novel mutations in Niemann-Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1.

The two known complementation groups of Niemann-Pick Type C disease, NPC1 and NPC2, result from non-allelic protein defects. Both the NPC1 and NPC2 (HE1) gene products are intimately involved in cholesterol and glycolipid trafficking and/or transport. We describe mutation analysis on samples from 143 unrelated affected NPC patients using conformation sensitive gel electrophoresis and DNA sequencing as the primary mutation screening methods for NPC1 and NPC2, respectively. These methods are robust, sensitive, and do not require any specialized laboratory equipment. Analyses identified two NPC1 mutations for 115 (80.4%) patients, one NPC1 mutation for 10 (7.0%) patients, two NPC2 mutations for five (3.5%) patients, one NPC2 mutation for one (0.7%) patient, and no mutations for 12 (8.4%) patients. Thus, mutations were identified on 251 of 286 (88%) disease alleles, including 121 different mutations (114 in NPC1 and seven in NPC2), 58 of which are previously unreported. The most common NPC1 mutation, I1061T, was detected on 18% of NPC alleles. Other NPC1 mutations were mostly private, missense mutations located throughout the gene with clustering in the cysteine-rich luminal domain. Correlation with biochemical data suggests classification of several mutations as severe and others as moderate or variable. The region between amino acids 1038 and 1253, which shares 35% identity with Patched 1, appears to be a hot spot for mutations. Additionally, a high percentage of mutations were located at amino acids identical to the NPC1 homolog, NPC1L1. Biochemical complementation analysis of cases negative for mutations revealed a high percentage of equivocal results where the complementation group appeared to be non-NPC1 and non-NPC2. This raises the possibilities of an additional NPC complementation group(s) or non-specificity of the biochemical testing for NPC. These caveats must be considered when offering mutation testing as a clinical service.