The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

MLPA-based approach for initial and simultaneous detection of GBA deletions and recombinant alleles in patients affected by Gaucher Disease.

The chromosomal region, in which the GBA gene is located, is structurally subject to misalignments, reciprocal and nonreciprocal homologous recombination events, leading to structural defects such as deletions, duplications and gene-pseudogene complex rearrangements causing Gaucher Disease (GD). Interestingly deletions and duplications, belonging to the heterogeneous group of structural defects collectively termed Copy Number Variations (CNVs), together with gene-pseudogene complex rearrangements represent the main cause of pitfalls in GD mutational analysis. In the present study, we set up and validate a Multiplex Ligation-dependent Probe Amplification (MLPA)-based approach to simultaneously investigate the potential occurrence of CNVs and complex rearrangements in 8 unrelated GD patients who had still not-well-characterized or uncharacterized alleles. The findings allowed us to complete the mutational analysis in 4 patients, identifying a rare deletion (g.-3100_+834del3934) and 2 novel recombinant alleles (g.4356_7031conJ03060.1:g.2544_4568; g.1942_7319conJ03060.1:g.1092_4856). These results demonstrate the diagnostic usefulness of MLPA in the detection of GBA deletions and recombinations. In addition, MLPA findings have also served as a basis for developing molecular approaches to precisely pinpoint the breakpoints and characterize the underlying mechanism of copy number variations.

Intragenic KANSL1 mutations and chromosome 17q21.31 deletions: broadening the clinical spectrum and genotype-phenotype correlations in a large cohort of patients.

BACKGROUND: The 17q21.31 deletion syndrome phenotype can be caused by either chromosome deletions or point mutations in the KANSL1 gene. To date, about 60 subjects with chromosome deletion and 4 subjects with point mutation in KANSL1 have been reported. Prevalence of chromosome deletions compared with point mutations, genotype-phenotype correlations and phenotypic variability have yet to be fully clarified. METHODS: We report genotype-phenotype correlations in 27 novel subjects with 17q21.31 deletion and in 5 subjects with KANSL1 point mutation, 3 of whom were not previously reported. RESULTS: The prevalence of chromosome deletion and KANSL1 mutation was 83% and 17%, respectively. All patients had similar clinical features, with the exception of macrocephaly, which was detected in 24% of patients with the deletion and 60% of those with the point mutation, and congenital heart disease, which was limited to 35% of patients with the deletion. A remarkable phenotypic variability was observed in both categories, mainly with respect to the severity of ID. Cognitive function was within normal parameters in one patient in each group. Craniosynostosis, subependymal heterotopia and optic nerve hypoplasia represent new component manifestations. CONCLUSIONS: In KANSL1 haploinsufficiency syndrome, chromosome deletions are greatly prevalent compared with KANSL1 mutations. The latter are sufficient in causing the full clinical phenotype. The degree of intellectual disability (ID) appears to be milder than expected in a considerable number of subjects with either chromosome deletion or KANSL1 mutation. Striking clinical criteria for enrolling patients into KANSL1 analysis include speech delay, distinctive facial dysmorphism, macrocephaly and friendly behaviour.

Lethal Capecitabine Toxicity in Patients With Complete Dihydropyrimidine Dehydrogenase Deficiency Due to Ultra-Rare DPYD Variants.

A DPD deficiency should be considered in case of severe toxicity even in the absence of common risk variants in DPYD.

Deletions within COL11A1 in Type 2 stickler syndrome detected by multiplex ligation-dependent probe amplification (MLPA).

BACKGROUND: COL11A1 is a large complex gene around 250 kb in length and consisting of 68 exons. Pathogenic mutations in the gene can result in Stickler syndrome, Marshall syndrome or Fibrochondrogenesis. Many of the mutations resulting in either Stickler or Marshall syndrome alter splice sites and result in exon skipping, which because of the exon structure of collagen genes usually leaves the message in-frame. The mutant protein then exerts a dominant negative effect as it co-assembles with other collagen gene products. To date only one large deletion of 40 kb in the COL11A1, which was detected by RT-PCR, has been characterized. However, commonly used screening protocols, utilizing genomic amplification and exon sequencing, are unlikely to detect such large deletions. Consequently the frequency of this type of mutation is unknown. CASE PRESENTATIONS: We have used Multiplex Ligation-Dependent Probe Amplification (MLPA) in conjunction with exon amplification and sequencing, to analyze patients with clinical features of Stickler syndrome, and have detected six novel deletions that were not found by exon sequencing alone. CONCLUSION: Exon deletions appear to represent a significant proportion of type 2 Stickler syndrome. This observation was previously unknown and so diagnostic screening of COL11A1 should include assays capable of detecting both large and small deletions, in addition to exon sequencing.

A multiplex assay to rapidly exclude HLA-DQ2.5 and HLA-DQ8 expression in patients at risk for celiac disease.

BACKGROUND: Celiac disease (CD) is an inflammatory disorder of the small intestine induced by gluten ingestion. CD has a strong genetic association with human leukocyte antigen (HLA)-DQ2.5 and HLA-DQ8. The absence of HLA-DQ2.5 and HLA-DQ8 has a strong negative predictive value for CD. Genetic screening of HLA-DQ2.5 and HLA-DQ8 in patients at risk is of great value. METHODS: We designed, developed, and validated a multiplex assay based on multiplex ligation-dependent probe amplification (MLPA) technology, allowing the simultaneous detection of DQA1*05-DQB1*02, encoding HLA-DQ2.5, and DQA1*03-DQB1*03:02, encoding HLA-DQ8. The amplified products were separated and identified using capillary electrophoresis. RESULTS: When compared with a polymerase chain reaction followed by single-strand conformation polymorphism/ heteroduplex analysis, one discrepancy was found. Sequencing analysis showed that the developed MLPA assay result was correct. Furthermore, we demonstrated that the MLPA method is able to distinguish between the heterozygote and homozygote expression of HLA-DQ2.5 or HLA-DQ8. CONCLUSIONS: This study shows that it is possible to rapidly and accurately screen for the absence of HLA-DQ2.5 and HLA-DQ8 using MLPA, excluding patients at risk for CD for further serological or histological follow-up. In addition, MLPA might be an accurate tool to screen for other specific HLA types in the context of disease association in a diagnostic laboratory setting.

Diagnostic analysis of the highly complex OPN1LW/OPN1MW gene cluster using long-read sequencing and MLPA.

Pathogenic variants in the OPN1LW/OPN1MW gene cluster are causal for a range of mild to severe visual impairments with color deficiencies. The widely utilized short-read next-generation sequencing (NGS) is inappropriate for the analysis of the OPN1LW/OPN1MW gene cluster and many patients with pathogenic variants stay underdiagnosed. A diagnostic genetic assay was developed for the OPN1LW/OPN1MW gene cluster, consisting of copy number analysis via multiplex ligation-dependent probe amplification and sequence analysis via long-read circular consensus sequencing. Performance was determined on 50 clinical samples referred for genetic confirmation of the clinical diagnosis (n = 43) or carrier status analysis (n = 7). A broad range of pathogenic haplotypes were detected, including deletions, hybrid genes, single variants and combinations of variants. The developed genetic assay for the OPN1LW/OPN1MW gene cluster is a diagnostic test that can detect both structural and nucleotide variants with a straightforward analysis, improving diagnostic care of patients with visual impairment.

Characterization of a novel transcript of the EHMT1 gene reveals important diagnostic implications for Kleefstra syndrome.

The core phenotype of Kleefstra syndrome (KS) is characterized by intellectual disability, childhood hypotonia, and a characteristic facial appearance. This can be caused by either submicroscopic 9q34 deletions or loss of function mutations of the EHMT1 gene. Remarkably, in three patients with a clinical suspicion of KS, molecular cytogenetic analysis revealed an interstitial 9q34 microdeletion proximal to the coding region of the EHMT1 gene based on the NM_ 024757.3 transcript. Because we found a mono-allelic EHMT1 transcript suggestive for haploinsufficiency of EHMT1 in two of these patients tested, we hypothesized that a deletion of regulatory elements or so far unknown coding sequences in the 5' region of the EHMT1 gene, might result in a phenotype compatible with KS. We further characterized the molecular content of deletions proximal to the transcript NM_ 024757.3 and confirmed presence of a novel predicted open reading frame comprising 27 coding exons (NM_ 024757.4). Further analysis showed that all three deletions included the presumed novel first exon of the EHMT1 gene. Subsequent testing of 75 individuals without previously detectable EHMT1 aberrations showed one additional case with a deletion comprising only this 5' part of the gene. These results have important implications for the genetic screening of KS and for studies of the functional significance of EHMT1.

Identification of SPRED1 deletions using RT-PCR, multiplex ligation-dependent probe amplification and quantitative PCR.

Legius syndrome, is a recently identified autosomal dominant disorder caused by loss of function mutations in the SPRED1 gene, with individuals mainly presenting with multiple café-au-lait macules (CALM), freckling and macrocephaly. So far, only SPRED1 point mutations have been identified as the cause of this syndrome. To determine if copy number changes (CNCs) are a cause of Legius syndrome, we have used a Multiplex Ligation-dependent Probe Amplification (MLPA) assay covering all SPRED1 exons in a cohort of 510 NF1-negative patients presenting with multiple CALMs with or without freckling, but no other NF1 diagnostic signs. Four different deletions were identified by MLPA and confirmed by quantitative PCR, reverse transcriptase PCR and/or array CGH: a deletion of exon 1 and the SPRED1 promoter region in a proband and two first-degree relatives; a deletion of the entire SPRED1 gene in a sporadic patient; a deletion of exon 2-6 in a proband and her father; and an ∼6.6 Mb deletion on chromosome 15 that spans SPRED1 in a sporadic patient. Deletions account for ∼10% of the 40 detected SPRED1 mutations in this cohort of 510 individuals. These results indicate the need for dosage analysis to complement sequencing-based SPRED1 mutation analyses.

Preemptive screening of DPYD as part of clinical practice: high prevalence of a novel exon 4 deletion in the Finnish population.

Capecitabine is a fluoropyrimidine that is widely used as a cancer drug for the treatment of patients with a variety of cancers. Unfortunately, early onset, severe or life-threatening toxicity is observed in 19-32% of patients treated with capecitabine and 5FU. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the degradation of 5FU and a DPD deficiency has been shown to be a major determinant of severe fluoropyrimidine-associated toxicity. DPD is encoded by the DPYD gene and some of the identified variants have been described to cause DPD deficiency. Preemptive screening for DPYD gene alterations enables the identification of DPD-deficient patients before administering fluoropyrimidines. In this article, we describe the application of upfront DPD screening in Finnish patients, as a part of daily clinical practice, which was based on a comprehensive DPYD gene analysis, measurements of enzyme activity and plasma uracil concentrations. Almost 8% of the patients (13 of 167 patients) presented with pathogenic DPYD variants causing DPD deficiency. The DPD deficiency in these patients was further confirmed via analysis of the DPD activity and plasma uracil levels. Interestingly, we identified a novel intragenic deletion in DPYD which includes exon 4 in four patients (31% of patients carrying a pathogenic variant). The high prevalence of the exon 4 deletion among Finnish patients highlights the importance of full-scale DPYD gene analysis. Based on the literature and our own experience, genotype preemptive screening should always be used to detect DPD-deficient patients before fluoropyrimidine therapy.

SMN1 Duplications Are Associated With Progressive Muscular Atrophy, but Not With Multifocal Motor Neuropathy and Primary Lateral Sclerosis.

OBJECTIVE: To assess the association between copy number (CN) variation in the survival motor neuron (SMN) locus and multifocal motor neuropathy (MMN), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS) susceptibility and to determine the association of SMN1 and SMN2 CN with MMN, PMA, and PLS disease course. METHODS: In this monocenter study, we used multiplex ligation-dependent probe amplification to determine SMN1 and SMN2 CN in Dutch patients with MMN, PMA, and PLS and controls. We stratified clinical parameters for SMN1 and SMN2 CN. We analyzed SMN1 and SMN2 exons 1-6, intron 6, and exon 8 CN to study the genetic architecture of SMN1 duplications. RESULTS: SMN1 and SMN2 CN were determined in 132 patients with MMN, 150 patients with PMA, 104 patients with PLS, and 956 control subjects. MMN and PLS were not associated with CN variation in SMN1 or SMN2. By contrast, patients with PMA more often than controls carried SMN1 duplications (≥3 SMN1 copies, 12.0% vs 5.0%, odds ratio 2.69 (1.43-4.91), p 0.0020). SMN1 and SMN2 CN status was not associated with MMN, PLS, or PMA disease course. In case of SMN1 exon 7 duplications, exons 1-6, exon 8, and introns 6 and 7 were also duplicated, suggesting full SMN1 duplications. CONCLUSIONS: SMN1 duplications are associated with PMA, but not with PLS and MMN. SMN1 duplications in PMA are balanced duplications. The results of this study highlight the primary effect of altered SMN CN on lower motor neurons.

Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity.

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21-23 was observed. In five patients a deep intronic mutation c.1129-5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129-5967 to c.1129-5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129-5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959-51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129-5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129-5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing.

Molecular profiling of invasive breast cancer by multiplex ligation-dependent probe amplification-based copy number analysis of tumor suppressor and oncogenes.

Several oncogenes and tumor-suppressor genes have been shown to be implicated in the development, progression and response to therapy of invasive breast cancer. The phenotypic uniqueness (and thus the heterogeneity of clinical behavior) among patients' tumors may be traceable to the underlying variation in gene copy number of these genes. To obtain a more complete view of gene copy number changes and their relation to phenotype, we analyzed 20 breast cancer-related genes in 104 invasive breast cancers with the use of multiplex ligation-dependent probe amplification (MLPA). We identified MYC gene amplification in 48% of patients, PRDM14 in 34%, topoisomerase IIalpha (TOP2A) in 32%, ADAM9 in 32%, HER2 in 28%, cyclin D1 (CCND1) in 26%, EMSY in 25%, IKBKB in 21%, AURKA in 17%, FGFR1 in 17%, estrogen receptor alpha (ESR1) in 16%, CCNE1 in 12% and EGFR in 9% of patients. There was a significant correlation between the number of amplified genes and the histological grade and mitotic index of the tumor. Gene amplifications of EGFR, CCNE1 and HER2 were negatively associated with estrogen receptor status whereas FGFR1, ADAM9, IKBKB and TOP2A revealed a positive association. Amplifications of ESR1, PRDM14, MYC and HER2 were associated with a high mitotic index, and PRDM14 and HER2 amplifications with high histological grade. MYC amplification was detected more frequently in ductal tumors and high-level MYC amplifications were significantly associated with large tumor size. HER2/MYC, HER2/CCNE1 and EGFR/MYC co-amplified tumors were significantly larger than tumors with either of these amplifications. Gene loss occurred most frequently in E-cadherin (CDH1) (20%) and FGFR1 (10%). In conclusion, MLPA analysis with this 'breast cancer kit' allowed to simultaneously assess copy numbers of 20 important breast cancer genes, providing an overview of the most frequent (co)amplifications as well as interesting phenotypic correlations, and thereby data on the potential importance of these genes in breast cancer.

Intragenic and structural variation in the SMN locus and clinical variability in spinal muscular atrophy.

Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than SMN2 copy number in the SMN locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1-4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the SMN locus. SMN1, SMN2 and NAIP gene copy number were determined by multiplex ligation-dependent probe amplification. SMN2 gene variant analysis was performed using Sanger sequencing and RNA expression analysis of SMN by droplet digital polymerase chain reaction. We identified SMN1-SMN2 hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within SMN1 and SMN2 genes. This indicates that SMN2 copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic SMN2 variants, which were associated with disease severity in individual cases. There are no indications that NAIP1 gene copy number or sequence variants add value in addition to SMN2 copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal SMN2 copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the SMN locus. SMN2 gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of SMN1 and SMN2 gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices.

Analysis of severely affected patients with dihydropyrimidine dehydrogenase deficiency reveals large intragenic rearrangements of DPYD and a de novo interstitial deletion del(1)(p13.3p21.3).

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14-16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3-1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.

High frequency of large genomic deletions in the PCCA gene causing propionic acidemia.

Mutations in either the PCCA or PCCB genes are responsible for propionic acidemia (PA), one of the most frequent organic acidemias inherited in autosomal recessive fashion. Most of the mutations detected to date in both genes are missense. In the case of PCCA deficient patients, a high number of alleles remain uncharacterized, some of them suspected to carry an exonic deletion. We have now employed multiplex ligation probe amplification (MLPA) and long-PCR in some cases to screen for genomic rearrangements in the PCCA gene in 20 patients in whom standard mutation detection techniques had failed to complete genotype analysis. Eight different deletions were found, corresponding to a frequency of 21.3% of the total PCCA alleles genotyped at our center. Two of the exonic deletions were frequent, one involving exons 3-4 and another exon 23 although in the first case two different chromosomal breakpoints were identified. Absence of exons 3 and 4 which is also the consequence of the novel splicing mutation c.231+1g>c present in two patients, presumably results in an in-frame deletion covering 39 aminoacids, which was expressed in a eukaryotic system confirming its pathogenicity. This work describes for the first time the high frequency of large genomic deletions in the PCCA gene, which could be due to the characteristics of the PCCA gene structure and its abundance in intronic repetitive elements. Our data underscore the need of using gene dosage analysis to complement routine genetic analysis in PCCA patients.

Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome.

CONTEXT: Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation. OBJECTIVE: To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients. DESIGN, SETTING, AND PARTICIPANTS: In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis. MAIN OUTCOME MEASURES: Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations. RESULTS: Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS. CONCLUSIONS: A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1.

The frequency of SMN gene variants lacking exon 7 and 8 is highly population dependent.

Spinal Muscular Atrophy (SMA) is a disorder characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. In the majority of cases, SMA is caused by the homozygous absence of the SMN1 gene. The disease severity of SMA is strongly influenced by the copy number of the closely related SMN2 gene. In addition, an SMN variant lacking exons 7 and 8 has been reported in 8% and 23% of healthy Swedish and Spanish individuals respectively. We tested 1255 samples from the 1000 Genomes Project using a new version of the multiplex ligation-dependent probe amplification (MLPA) P021 probemix that covers each SMN exon. The SMN variant lacking exons 7 and 8 was present in up to 20% of individuals in several Caucasian populations, while being almost completely absent in various Asian and African populations. This SMN1/2Δ7-8 variant appears to be derived from an ancient deletion event as the deletion size is identical in 99% of samples tested. The average total copy number of SMN1, SMN2 and the SMN1/2Δ7-8 variant combined was remarkably comparable in all populations tested, ranging from 3.64 in Asian to 3.75 in African samples.

Integrated CYP2D6 interrogation for multiethnic copy number and tandem allele detection.

AIM: To comprehensively interrogate CYP2D6 by integrating genotyping, copy number analysis and novel strategies to identify CYP2D6*36 and characterize CYP2D6 duplications. METHODS: Genotyping of 16 CYP2D6 alleles, multiplex ligation-dependent probe amplification (MLPA) and CYP2D6*36 and duplication allele-specific genotyping were performed on 427 African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals. RESULTS: A novel PCR strategy determined that almost half of all CYP2D6*10 (100C>T) alleles are actually *36 (isolated or in tandem with *10) and all identified duplication alleles were characterized. Integrated results from all testing platforms enabled the refinement of genotype frequencies across all studied populations. CONCLUSION: The polymorphic CYP2D6 gene requires comprehensive interrogation to characterize allelic variation across ethnicities, which was enabled in this study by integrating multiplexed genotyping, MLPA copy number analysis, novel PCR strategies and duplication allele-specific genotyping.