Detection of PTCH1 Copy-Number Variants in Mosaic Basal Cell Nevus Syndrome.

Basal cell nevus syndrome (BCNS) is an inherited disorder characterized mainly by the development of basal cell carcinomas (BCCs) at an early age. BCNS is caused by heterozygous small-nucleotide variants (SNVs) and copy-number variants (CNVs) in the Patched1 (PTCH1) gene. Genetic diagnosis may be complicated in mosaic BCNS patients, as accurate SNV and CNV analysis requires high-sensitivity methods due to possible low variant allele frequencies. We compared test outcomes for PTCH1 CNV detection using multiplex ligation-probe amplification (MLPA) and digital droplet PCR (ddPCR) with samples from a BCNS patient heterozygous for a PTCH1 CNV duplication and the patient's father, suspected to have a mosaic form of BCNS. ddPCR detected a significantly increased PTCH1 copy-number ratio in the index patient's blood, and the father's blood and tissues, indicating that the father was postzygotic mosaic and the index patient inherited the CNV from him. MLPA only detected the PTCH1 duplication in the index patient's blood and in hair and saliva from the mosaic father. Our data indicate that ddPCR more accurately detects CNVs, even in low-grade mosaic BCNS patients, which may be missed by MLPA. In general, quantitative ddPCR can be of added value in the genetic diagnosis of mosaic BCNS patients and in estimating the recurrence risk for offspring.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

Whole Exome Sequencing Is the Preferred Strategy to Identify the Genetic Defect in Patients With a Probable or Possible Mitochondrial Cause.

Mitochondrial disorders, characterized by clinical symptoms and/or OXPHOS deficiencies, are caused by pathogenic variants in mitochondrial genes. However, pathogenic variants in some of these genes can lead to clinical manifestations which overlap with other neuromuscular diseases, which can be caused by pathogenic variants in non-mitochondrial genes as well. Mitochondrial pathogenic variants can be found in the mitochondrial DNA (mtDNA) or in any of the 1,500 nuclear genes with a mitochondrial function. We have performed a two-step next-generation sequencing approach in a cohort of 117 patients, mostly children, in whom a mitochondrial disease-cause could likely or possibly explain the phenotype. A total of 86 patients had a mitochondrial disorder, according to established clinical and biochemical criteria. The other 31 patients had neuromuscular symptoms, where in a minority a mitochondrial genetic cause is present, but a non-mitochondrial genetic cause is more likely. All patients were screened for pathogenic variants in the mtDNA and, if excluded, analyzed by whole exome sequencing (WES). Variants were filtered for being pathogenic and compatible with an autosomal or X-linked recessive mode of inheritance in families with multiple affected siblings and/or consanguineous parents. Non-consanguineous families with a single patient were additionally screened for autosomal and X-linked dominant mutations in a predefined gene-set. We identified causative pathogenic variants in the mtDNA in 20% of the patient-cohort, and in nuclear genes in 49%, implying an overall yield of 68%. We identified pathogenic variants in mitochondrial and non-mitochondrial genes in both groups with, obviously, a higher number of mitochondrial genes affected in mitochondrial disease patients. Furthermore, we show that 31% of the disease-causing genes in the mitochondrial patient group were not included in the MitoCarta database, and therefore would have been missed with MitoCarta based gene-panels. We conclude that WES is preferable to panel-based approaches for both groups of patients, as the mitochondrial gene-list is not complete and mitochondrial symptoms can be secondary. Also, clinically and genetically heterogeneous disorders would require sequential use of multiple different gene panels. We conclude that WES is a comprehensive and unbiased approach to establish a genetic diagnosis in these patients, able to resolve multi-genic disease-causes.

Genetic defects in mtDNA-encoded protein translation cause pediatric, mitochondrial cardiomyopathy with early-onset brain disease.

This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.

Selection and Characterization of Palmitic Acid Responsive Patients with an OXPHOS Complex I Defect.

Mitochondrial disorders are genetically and clinically heterogeneous, mainly affecting high energy-demanding organs due to impaired oxidative phosphorylation (OXPHOS). Currently, effective treatments for OXPHOS defects, with complex I deficiency being the most prevalent, are not available. Yet, clinical practice has shown that some complex I deficient patients benefit from a high-fat or ketogenic diet, but it is unclear how these therapeutic diets influence mitochondrial function and more importantly, which complex I patients could benefit from such treatment. Dietary studies in a complex I deficient patient with exercise intolerance showed increased muscle endurance on a high-fat diet compared to a high-carbohydrate diet. We performed whole-exome sequencing to characterize the genetic defect. A pathogenic homozygous p.G212V missense mutation was identified in the TMEM126B gene, encoding an early assembly factor of complex I. A complementation study in fibroblasts confirmed that the p.G212V mutation caused the complex I deficiency. The mechanism turned out to be an incomplete assembly of the peripheral arm of complex I, leading to a decrease in the amount of mature complex I. The patient clinically improved on a high-fat diet, which was supported by the 25% increase in maximal OXPHOS capacity in TMEM126B defective fibroblast by the saturated fatty acid palmitic acid, whereas oleic acid did not have any effect in those fibroblasts. Fibroblasts of other patients with a characterized complex I gene defect were tested in the same way. Patient fibroblasts with complex I defects in NDUFS7 and NDUFAF5 responded to palmitic acid, whereas ACAD9, NDUFA12, and NDUFV2 defects were non-responding. Although the data are too limited to draw a definite conclusion on the mechanism, there is a tendency that protein defects involved in early assembly complexes, improve with palmitic acid, whereas proteins defects involved in late assembly, do not. Our data show at a clinical and biochemical level that a high fat diet can be beneficial for complex I patients and that our cell line assay will be an easy tool for the selection of patients, who might potentially benefit from this therapeutic diet.

Novel SLC25A32 mutation in a patient with a severe neuromuscular phenotype.

In a 51-year-old patient of consanguineous parents with a severe neuromuscular phenotype of early-onset ataxia, myoclonia, dysarthria, muscle weakness and exercise intolerance, exome sequencing revealed a novel homozygous variant (c.-264_31delinsCTCACAAATGCTCA) in the mitochondrial FAD-transporter gene SLC25A32. Flavin adenine dinucleotide (FAD) is an essential co-factor for many mitochondrial enzymes and impaired mitochondrial FAD-transport was supported by a reduced oxidative phosphorylation complex II activity in the patient's muscle, decreased ATP production in fibroblasts, and a deficiency of mitochondrial FAD-dependent enzymes. Clinically, the patient showed improvement upon riboflavin treatment, which is a precursor of FAD. Our results confirm the recently reported case of SLC25A32 as a cause of riboflavin-responsive disease. Our patient showed a more severe clinical phenotype compared with the reported patient, corresponding with the (most likely) complete absence of the SLC25A32-encoding MFT (Mitochondrial Folate Transporter) protein.

Rapid Resolution of Blended or Composite Multigenic Disease in Infants by Whole-Exome Sequencing.

Whole-exome sequencing identified multiple genetic causes in 2 infants with heterogeneous disease. Three gene defects in the first patient explained all symptoms, but manifestations were overlapping (blended phenotype). Two gene defects in the second patient explained nonoverlapping symptoms (composite phenotype). Whole-exome sequencing rapidly and comprehensively resolves heterogeneous genetic disease.

Specific MRI Abnormalities Reveal Severe Perrault Syndrome due to CLPP Defects.

In establishing a genetic diagnosis in heterogeneous neurological disease, clinical characterization and whole exome sequencing (WES) go hand-in-hand. Clinical data are essential, not only to guide WES variant selection and define the clinical severity of a genetic defect but also to identify other patients with defects in the same gene. In an infant patient with sensorineural hearing loss, psychomotor retardation, and epilepsy, WES resulted in identification of a novel homozygous CLPP frameshift mutation (c.21delA). Based on the gene defect and clinical symptoms, the diagnosis Perrault syndrome type 3 (PRLTS3) was established. The patient's brain-MRI revealed specific abnormalities of the subcortical and deep cerebral white matter and the middle blade of the corpus callosum, which was used to identify similar patients in the Amsterdam brain-MRI database, containing over 3000 unclassified leukoencephalopathy cases. In three unrelated patients with similar MRI abnormalities the CLPP gene was sequenced, and in two of them novel missense mutations were identified together with a large deletion that covered part of the CLPP gene on the other allele. The severe neurological and MRI abnormalities in these young patients were due to the drastic impact of the CLPP mutations, correlating with the variation in clinical manifestations among previously reported patients. Our data show that similarity in brain-MRI patterns can be used to identify novel PRLTS3 patients, especially during early disease stages, when only part of the disease manifestations are present. This seems especially applicable to the severely affected cases in which CLPP function is drastically affected and MRI abnormalities are pronounced.

Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.