Quantifying constraint in the human mitochondrial genome.

Mitochondrial DNA (mtDNA) has an important yet often overlooked role in health and disease. Constraint models quantify the removal of deleterious variation from the population by selection and represent powerful tools for identifying genetic variation that underlies human phenotypes1-4. However, nuclear constraint models are not applicable to mtDNA, owing to its distinct features. Here we describe the development of a mitochondrial genome constraint model and its application to the Genome Aggregation Database (gnomAD), a large-scale population dataset that reports mtDNA variation across 56,434 human participants5. Specifically, we analyse constraint by comparing the observed variation in gnomAD to that expected under neutrality, which was calculated using a mtDNA mutational model and observed maximum heteroplasmy-level data. Our results highlight strong depletion of expected variation, which suggests that many deleterious mtDNA variants remain undetected. To aid their discovery, we compute constraint metrics for every mitochondrial protein, tRNA and rRNA gene, which revealed a range of intolerance to variation. We further characterize the most constrained regions within genes through regional constraint and identify the most constrained sites within the entire mitochondrial genome through local constraint, which showed enrichment of pathogenic variation. Constraint also clustered in three-dimensional structures, which provided insight into functionally important domains and their disease relevance. Notably, we identify constraint at often overlooked sites, including in rRNA and noncoding regions. Last, we demonstrate that these metrics can improve the discovery of deleterious variation that underlies rare and common phenotypes.

The Australian Genomics Mitochondrial Flagship: A National Program Delivering Mitochondrial Diagnoses.

PURPOSE: Families living with mitochondrial diseases (MD) often endure prolonged diagnostic journeys and invasive testing, yet many remain without a molecular diagnosis. The Australian Genomics Mitochondrial flagship, comprising clinicians, diagnostic, and research scientists, conducted a prospective national study to identify the diagnostic utility of singleton genomic sequencing using blood samples. METHODS: 140 children and adults living with suspected MD were recruited using modified Nijmegen criteria (MNC) and randomized to either exome + mtDNA sequencing (ES+mtDNAseq) or genome sequencing (GS). RESULTS: Diagnostic yield was 55% (n=77) with variants in nuclear (n=37) and mtDNA (n=18) MD genes, as well as phenocopy genes (n=22). A nuclear gene etiology was identified in 77% of diagnoses, irrespective of disease onset. Diagnostic rates were higher in pediatric-onset (71%) than adult-onset (31%) cases, and comparable in children with non-European (78%) versus European (67%) ancestry. For children, higher MNC scores correlated with increased diagnostic yield and fewer diagnoses in phenocopy genes. Additionally, three adult patients had a mtDNA deletion discovered in skeletal muscle that was not initially identified in blood. CONCLUSION: Genomic sequencing from blood can simplify the diagnostic pathway for individuals living with suspected MD, especially those with childhood onset diseases and high MNC scores.

Correction: Reproductive genetic carrier screening for cystic fibrosis, fragile X syndrome, and spinal muscular atrophy in Australia: outcomes of 12,000 tests.

Zoe McDonald, BSc, was omitted from the list of article coauthors. Her name should have been included as the seventh author, following Clare Elizabeth Hunt. Her affiliation is Victorian Clinical Genetics Services, Parkville, Victoria, Australia. The authors regret the error.

Reproductive genetic carrier screening for cystic fibrosis, fragile X syndrome, and spinal muscular atrophy in Australia: outcomes of 12,000 tests.

PurposeTo describe our experience of offering simultaneous genetic carrier screening for cystic fibrosis (CF), fragile X syndrome (FXS), and spinal muscular atrophy (SMA).MethodsCarrier screening is offered through general practice, obstetrics, fertility, and genetics settings before or in early pregnancy. Carriers are offered genetic counseling with prenatal/preimplantation genetic diagnosis available to those at increased risk.ResultsScreening of 12,000 individuals revealed 610 carriers (5.08%; 1 in 20): 342 CF, 35 FXS, 241 SMA (8 carriers of 2 conditions), approximately 88% of whom had no family history. At least 94% of CF and SMA carriers' partners were tested. Fifty couples (0.42%; 1 in 240) were at increased risk of having a child with one of the conditions (14 CF, 35 FXS, and 1 SMA) with 32 pregnant at the time of testing. Of these, 26 opted for prenatal diagnosis revealing 7 pregnancies affected (4 CF, 2 FXS, 1 SMA).ConclusionThe combined affected pregnancy rate is comparable to the population risk for Down syndrome, emphasizing the need to routinely offer carrier screening. The availability of appropriate genetic counseling support and a collaborative approach between laboratory teams, genetics services, health professionals offering screening, and support organizations is essential.

A prospective evaluation of whole-exome sequencing as a first-tier molecular test in infants with suspected monogenic disorders.

PURPOSE: To prospectively evaluate the diagnostic and clinical utility of singleton whole-exome sequencing (WES) as a first-tier test in infants with suspected monogenic disease. METHODS: Singleton WES was performed as a first-tier sequencing test in infants recruited from a single pediatric tertiary center. This occurred in parallel with standard investigations, including single- or multigene panel sequencing when clinically indicated. The diagnosis rate, clinical utility, and impact on management of singleton WES were evaluated. RESULTS: Of 80 enrolled infants, 46 received a molecular genetic diagnosis through singleton WES (57.5%) compared with 11 (13.75%) who underwent standard investigations in the same patient group. Clinical management changed following exome diagnosis in 15 of 46 diagnosed participants (32.6%). Twelve relatives received a genetic diagnosis following cascade testing, and 28 couples were identified as being at high risk of recurrence in future pregnancies. CONCLUSIONS: This prospective study provides strong evidence for increased diagnostic and clinical utility of singleton WES as a first-tier sequencing test for infants with a suspected monogenic disorder. Singleton WES outperformed standard care in terms of diagnosis rate and the benefits of a diagnosis, namely, impact on management of the child and clarification of reproductive risks for the extended family in a timely manner.Genet Med 18 11, 1090-1096.

Premature arthritis is a distinct type II collagen phenotype.

Mutations in the gene encoding type II collagen (COL2A1) give rise to a spectrum of phenotypes predominantly affecting cartilage and bone. These chondrodysplasias are typically characterized by disproportionately short stature, eye abnormalities, cleft palate, and hearing loss. It is less recognized that mutations in COL2A1 can also present as degenerative joint disease in the absence of any other phenotypic clues. We report 2 Australian families presenting with an isolated arthritis phenotype, segregating as a dominant trait affecting both large and small joints, prior to age 30 years. Sequencing of COL2A1 in the propositi revealed 2 sequence changes resulting in glycine substitutions in the triple-helical domain of type II collagen. We review the increasing evidence implicating COL2A1 mutations in individuals presenting with isolated degenerative joint disease, aiming to alert physicians who assess these patients to this possibility. The importance of finding a COL2A1 mutation in such patients lies in the subsequent ability to accurately assess recurrence risks, offer early (including prenatal) diagnosis, and provide information regarding the natural history of the condition. Most importantly, it enables at-risk individuals to be identified for implementation of preventative strategies (i.e., weight loss, joint-friendly exercise programs) and early ameliorative management of their condition.

Population screening and cascade testing for carriers of SMA.

Spinal muscular atrophy (SMA) is one of the most common autosomal-recessive diseases, caused by absence of both copies of the survival motor neuron 1 (SMN1) gene. Identification of SMA carriers has important implications for individuals with a family history and the general population. SMA carriers are completely healthy and most are unaware of their carrier status until they have an affected child. A total of 422 individuals have been studied to identify SMA carriers. This cohort included 117 parents of children homozygously deleted for SMN1 (94% were carriers and 6% had two copies of SMN1; of these individuals, two in seven had the '2+0' genotype, two in seven were normal but had children carrying a de novo deletion and three in seven were unresolved), 158 individuals with a significant family history of SMA (47% had one copy, 49% had two copies and 4% had three copies of SMN1) and 146 individuals with no family history of SMA (90% had two copies, 2% had one copy and 8% had three copies of SMN1). The SMA carrier frequency in the Australian population appears to be 1/49 and the frequency of two-copy SMN1 alleles and de novo deletion mutations are both at least 1.7%. A multimodal approach involving quantitative analysis, linkage analysis and genetic risk assessment (GRA), facilitates the resolution of SMA carrier status in individuals with a family history as well as individuals of the general population, providing couples with better choices in their family planning.

Identification of APC gene mutations in colorectal cancer using universal microarray-based combinatorial sequencing-by-hybridization.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited form of colorectal cancer, caused mostly by mutations in the APC gene. Due to the wide variety of mutations found and the large size of the APC gene, several methods of mutation detection are used, which can be time consuming and costly. Here we demonstrate a new method of mutation detection in the APC gene using an array-based approach termed combinatorial sequencing-by-hybridization (cSBH). In cSBH, a universal probe set is attached to a support and a second one is in solution. Two-probe ligation occurs when a DNA strand from the target PCR product consecutively anneals to both unlabeled array-bound and solution-phase dye-labeled probe, creating all target-complementary long-labeled probes attached to the surface. A standard array reader scores fluorescent signals at each array position. Cell lines and patient DNA with known APC gene mutations were analyzed using a cSBH-based HyChip trade mark product. Results show that this universal hexamer (6-mer) chip can successfully detect a range of mutations. Results are very robust for a continuous readout of 3.6 kb from a PCR target, with 99.97% accuracy on a single HyChip trade mark slide. cSBH is a fast, cost-efficient method for first stage mutation screening in the APC or any other gene.

Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH.

BACKGROUND & AIMS: Hyperplastic polyposis syndrome (HPS) is defined phenotypically with multiple, large and/or proximal hyperplastic polyps. There is no known germ-line predisposition. We aimed to characterize the clinicopathologic features of 38 patients with HPS and explore the role of germ-line mutations in the base excision repair genes MBD4 and MYH. METHODS: Utilizing clinical databases of The Royal Melbourne Hospital Bowel Cancer Surveillance Service and the Familial Cancer Clinic, 38 patients with HPS were recruited. The patients were analyzed for age at first diagnosis, features of hyperplastic polyposis, family histories of polyposis and colorectal cancer (CRC), coexisting adenomas, serrated adenomas, incidence of CRC, and microsatellite instability in the tumours. Mutation analysis of MBD4 and MYH were performed. RESULTS: Serrated adenomas were common (26%), and 19 (50%) of the 38 patients had a first-degree relative with CRC. Family history of HPS was uncommon, with only 2 cases found. Ten patients developed CRC, and 3 required surgery for polyposis. No pathogenic mutations in MBD4 were detected in the 27 patients tested, but 6 single nucleotide polymorphisms of uncertain functional significance were identified. Pathogenic biallelic MYH mutations were detected in 1 patient. CONCLUSIONS: Mutations in MBD4 are unlikely to be implicated in HPS; MYH mutations should be studied, especially when adenomas occur in the same patient. The clinical, histopathologic, and molecular findings of this study should contribute to our understanding of HPS and its relationship to the serrated neoplasia pathway.