Damien L. Bruno

Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing

Applying next-generation sequencing to 42 infants with mitochondrial disease highlights both the potential and the challenge of using this technology in clinical diagnosis. Getting to the Genetic Root of Mitochondrial Disease Next-generation DNA sequencing is being applied with great success in research settings to uncover new disease genes. Despite these successes, it is unclear how useful the technology will be for routine clinical diagnosis given the challenge of interpreting DNA variations in individual patients. In a new study, Calvo and colleagues apply next-generation sequencing to infants with mitochondrial disorders, a large collection of inherited diseases that are notoriously difficult to diagnose because of the multitude of candidate genes and the highly variable nature of the clinical presentation. First, the authors selected 42 unrelated infants with mitochondrial diseases that were refractory to standard clinical genetic testing. Then, for each child, they sequenced the DNA of the mitochondrial genome, the 100 genes previously linked to mitochondrial disease, and the ~1000 additional genes that are known to participate in mitochondrial biology. Of all the DNA differences present in the patients, the researchers prioritized those that were rare in the general population, predicted to disrupt protein function, and inherited in a recessive fashion. Such variants showed fivefold enrichment in the patients compared to that in healthy control individuals. In 10 patients (24%), firm molecular diagnoses were made in genes previously linked to mitochondrial diseases; 13 patients (31%) had prioritized recessive mutations in genes not previously linked to disease. For two of these genes, the authors were able to show that the mutations caused the mitochondrial disorder. These results suggest that next-generation sequencing may be able to provide a molecular diagnosis for ~25% of currently unsolved cases of infantile mitochondrial disease. An additional 25% of cases could be solved in the coming few years as more genes are formally proven to be linked to mitochondrial disease. The remaining 50% of patients in whom diagnosis was not possible underscores the challenge of interpreting DNA sequence data for clinical diagnosis. Nevertheless, the study by Calvo and colleagues will help to calibrate clinicians’ expectations regarding the diagnostic use of next-generation sequencing. Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. Although in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic use, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with more than 100 causal genes identified to date. We performed “MitoExome” sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Because patients and healthy control individuals harbored a comparable number of such heterozygous alleles, we could not prioritize dominant-acting genes. However, patients showed a fivefold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23 of 42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. Thirteen patients (31%) had mutations in nuclear genes not previously linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. The results underscore the potential and challenges of deploying NGS in clinical settings.

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases.

Extending the phenotype of recurrent rearrangements of 16p11.2: deletions in mentally retarded patients without autism and in normal individuals.

Array CGH (comparative genomic hybridization) screening of large patient cohorts with mental retardation and/or multiple congenital anomalies (MR/MCA) has led to the identification of a number of new microdeletion and microduplication syndromes. Recently, a recurrent copy number variant (CNV) at chromosome 16p11.2 was reported to occur in up to 1% of autistic patients in three large autism studies. In the screening of 4284 patients with MR/MCA with various array platforms, we detected 22 individuals (14 index patients and 8 family members) with deletions in 16p11.2, which are genomically identical to those identified in the autism studies. Though some patients shared a facial resemblance and a tendency to overweight, there was no evidence for a recognizable phenotype. Autism was not the presenting feature in our series. The assembled evidence indicates that recurrent 16p11.2 deletions are associated with variable clinical outcome, most likely arising from haploinsufficiency of one or more genes. The phenotypical spectrum ranges from MR and/or MCA, autism, learning and speech problems, to a normal phenotype.

Rapid, high throughput prenatal detection of aneuploidy using a novel quantitative method (MLPA)

Prenatal diagnosis of syndromes caused by chromosomal abnormality is a long established part of obstetric care in developed countries. In this area, there have been recent significant advances in the identification of high risk pregnancies using sophisticated serum analyte and ultrasound screening methods.1,2 For follow up diagnostic testing, karyotyping has provided the gold standard. This technology has remained essentially unchanged over 30 years, as no new technology has yet proven superior in terms of being able to detect such a wide range of abnormalities with the necessary precision. Nevertheless, molecular tests, such as fluorescent in situ hybridisation (FISH) 3 and short tandem repeat analysis,4 are now in common practice for the diagnosis of specific abnormalities. These adjunctive tests importantly decrease turnaround times from 1–2 weeks to 1–2 days. We assessed the performance of multiplex ligation dependent probe amplification (MLPA) as an alternative method for the detection of aneuploidy, which is by far the most common prenatal chromosome abnormality. This novel technique5 detects sequence dosage differences in a semi-quantitative manner and has many potential applications in diagnostic molecular genetics and cytogenetics. For example, a recent report describes its use for detection of large genomic deletions and duplications in the BRCA1 gene.6 This technology appears to have significant advantages in that it is extremely versatile in its applications, flexible in its target loci, highly automated, suitable for high throughput testing, efficient, and cost effective. Its application for aneuploidy detection has not been reported in a clinical setting. In order to assess the precision and robustness of the test, we conducted a prospective blind trial on 492 consecutive amniocentesis samples referred to our cytogenetics laboratory. ### Study design This study was designed to blind test amniocentesis samples prospectively. The samples were collected over a 4 month period as they were referred …

Further molecular and clinical delineation of co-locating 17p13.3 microdeletions and microduplications that show distinctive phenotypes

Background Chromosome 17p13.3 contains extensive repetitive sequences and is a recognised region of genomic instability. Haploinsufficiency of PAFAH1B1 (encoding LIS1) causes either isolated lissencephaly sequence or Miller–Dieker syndrome, depending on the size of the deletion. More recently, both microdeletions and microduplications mapping to the Miller–Dieker syndrome telomeric critical region have been identified and associated with distinct but overlapping phenotypes. Methods Genome-wide microarray screening was performed on 7678 patients referred with unexplained learning difficulties and/or autism, with or without other congenital abnormalities. Eight and five unrelated individuals, respectively, were identified with microdeletions and microduplications in 17p13.3. Results Comparisons with six previously reported microdeletion cases identified a 258 kb critical region, encompassing six genes including CRK (encoding Crk) and YWHAE (encoding 14-3-3ε). Clinical features included growth retardation, facial dysmorphism and developmental delay. Notably, one individual with only subtle facial features and an interstitial deletion involving CRK but not YWHAE suggested that a genomic region spanning 109 kb, encompassing two genes (TUSC5 and YWHAE), is responsible for the main facial dysmorphism phenotype. Only the microduplication phenotype included autism. The microduplication minimal region of overlap for the new and previously reported cases spans 72 kb encompassing a single gene, YWHAE. These genomic rearrangements were not associated with low-copy repeats and are probably due to diverse molecular mechanisms. Conclusions The authors further characterise the 17p13.3 microdeletion and microduplication phenotypic spectrum and describe a smaller critical genomic region allowing identification of candidate genes for the distinctive facial dysmorphism (microdeletions) and autism (microduplications) manifestations.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

Background: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Methods: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. Results: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as “potentially pathogenic”. Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Conclusions: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered “new syndromes” were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype–phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Molecular and clinical characterization of 25 individuals with exonic deletions of NRXN1 and comprehensive review of the literature

This study aimed to elucidate the observed variable phenotypic expressivity associated with NRXN1 (Neurexin 1) haploinsufficiency by analyses of the largest cohort of patients with NRXN1 exonic deletions described to date and by comprehensively reviewing all comparable copy number variants in all disease cohorts that have been published in the peer reviewed literature (30 separate papers in all). Assessment of the clinical details in 25 previously undescribed individuals with NRXN1 exonic deletions demonstrated recurrent phenotypic features consisting of moderate to severe intellectual disability (91%), severe language delay (81%), autism spectrum disorder (65%), seizures (43%), and hypotonia (38%). These showed considerable overlap with previously reported NRXN1‐deletion associated phenotypes in terms of both spectrum and frequency. However, we did not find evidence for an association between deletions involving the β‐isoform of neurexin‐1 and increased head size, as was recently published in four cases with a deletion involving the C‐terminus of NRXN1. We identified additional rare copy number variants in 20% of cases. This study supports a pathogenic role for heterozygous exonic deletions of NRXN1 in neurodevelopmental disorders. The additional rare copy number variants identified may act as possible phenotypic modifiers as suggested in a recent digenic model of neurodevelopmental disorders.

Pathogenic aberrations revealed exclusively by single nucleotide polymorphism (SNP) genotyping data in 5000 samples tested by molecular karyotyping

Background Several recent studies have demonstrated the use of single nucleotide polymorphism (SNP) arrays for the investigation of intellectual disability, developmental delay, autism or congenital abnormalities. In addition to LogR ‘copy number’ data, these arrays provide SNP genotyping data for gene level autozygosity mapping, estimating low levels of mosaicism, assessing long continuous stretches of homozygosity (LCSH), detection of uniparental disomy, and ‘autozygous’ regions. However, there remains little specific information on the clinical utility of this genotyping data. Methods Molecular karyotyping, using SNP array, was performed on 5000 clinical samples. Results Clinically significant ‘LogR neutral’ genotyping abnormalities were detected in 0.5% of cases. Among these were a single case of chimerism, 12 cases with low level chromosome mosaicism, and 11 cases with an LCSH associated with uniparental disomy. In addition, the genotyping data revealed several LCSH associated with clinically relevant ‘recessive type’ genetic defects. Conclusions These results demonstrate the utility of SNP genotyping data for detection of clinically significant abnormalities, including chimerism/mosaicism and recessive Mendelian disorders associated with autozygosity. The incidence of clinically significant low level mosaicism inferred from these cases suggests that this has hitherto been underestimated and chromosome mosaicism frequently occurs in the absence of indicative clinical features. The growing appreciation among clinicians and demand for SNP genotyping data poses significant challenges for the interpretation of LCSH, especially where there is no detailed phenotypic description to direct laboratory analysis. Finally, reporting of unexpected or hidden consanguinity revealed by SNP array analysis raises potential ethical and legal issues.

Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes

Iron-sulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called iron-sulfur or Fe-S proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203G>T, p.R68L) was identified via massively parallel sequencing of >1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other Fe-S proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of Fe-S proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.

High-throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy.

Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low‐resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation‐Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy‐number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty‐two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost‐effective, high‐throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage.