Patrick A. Lundquist

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.

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.

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 Barts) 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.

Development and validation of a comprehensive mutation and deletion detection assay for SDHB, SDHC, and SDHD

BACKGROUND Lack of sequencing validation and complexity of deletion testing hinder genetic diagnosis of SDH-associated paraganglioma/pheochromocytoma. METHODS We developed sequencing assays and multiplex ligation-dependent probe amplification (MLPA) deletion detection for SDHB, SDHC and SDHD. Clinical performance was validated on 141 blinded samples, previously tested at NIH. RESULTS Sequencing and deletion detection were highly reproducible and agreed with previous NIH results in 99.3% and 100%, respectively. CONCLUSIONS DNA sequencing combined with MLPA allows reliable and simplified genotyping of SDHB, SDHC and SDHD.

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.

Genetic testing for Niemann-Pick Type C disease

Niemann-Pick Type C (NPC) disease is an autosomal recessive lysosomal storage disorder that is characterized biochemically by sequestration of unesterified cholesterol and glycolipids in endosomal and/or lysosomal vesicles with consequent delay in cholesterol esterification. Clinical manifestations include progressive neurodegeneration, variable hepatosplenomegaly and vertical supranuclear gaze palsy. Death often occurs during childhood or early adulthood. NPC is a panethnic disorder with an estimated prevalence of 1 in 150,000. In approximately 95% of families the disease is linked to the NPC-1 gene at 18q11. This gene, isolated in 1997, contains 25 exons encoded by 3.9kb cDNA. Using multiplex PCR and CSGE to screen for mutations in NPC-1, we have tested genomic DNA from 53 unrelated affected individuals. Putative mutations were identified on 64 of 108 (59%) disease alleles and included 38 different DNA alterations located throughout the gene. Types of mutations included: missense (27), nonsense (1), frameshift (5), in-frame deletion (2) and splice site mutation (3). Recurrent mutations were 11061T (allele frequency 19/108, 18%), P237S (allele frequency 4/108, 3.7%), and dell271 (allele frequency 2/108, 1.9%). In addition to the putative mutations, 6 polymorphisms were identified. The finding of a high degree of mutation heterogeneity with many missense alterations creates difficulties for the clinical application of mutation testing in NPC. Although we can determine if missense alterations are in proposed functional domains or at sites conserved across species, we cannot be certain of the pathogenicity of such alterations. DNA alterations identified in affected patients can be used as linkage markers to determine carrier status of at risk relatives. However, the diagnosis in the affected individual must be confirmed by biochemical testing, and must include complementation analysis to establish that the defect is in NPC-1. Mutation analysis for individuals from the general population (e.g. partners of NPC carriers) is likely to result in significant dilemmas in interpretation of results. Funded by a grant from the Ara Parseghian Medical Research Foundation.

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.