A homozygous missense variant in type I keratin KRT25 causes autosomal recessive woolly hair.

Background: Woolly hair (WH) is a hair abnormality that is primarily characterised by tightly curled hair with abnormal growth.

Methods: In two unrelated consanguineous Pakistani families with non-syndromic autosomal recessive (AR) WH, homozygosity mapping and linkage analysis identified a locus within 17q21.1-q22, which contains the type I keratin gene cluster.

Rare A2ML1 variants confer susceptibility to otitis media.

A duplication variant within the middle ear-specific gene A2ML1 cosegregates with otitis media in an indigenous Filipino pedigree (LOD score = 7.5 at reduced penetrance) and lies within a founder haplotype that is also shared by 3 otitis-prone European-American and Hispanic-American children but is absent in non-otitis-prone children and >62,000 next-generation sequences. We identified seven additional A2ML1 variants in six otitis-prone children.

KIAA0586 is Mutated in Joubert Syndrome.

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by a distinctive mid-hindbrain malformation. JS is part of a group of disorders called ciliopathies based on their overlapping phenotypes and common underlying pathophysiology linked to primary cilium dysfunction. Biallelic mutations in one of 28 genes, all encoding proteins localizing to the primary cilium or basal body, can cause JS.

Joubert syndrome: a model for untangling recessive disorders with extreme genetic heterogeneity.

Background: Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterised by hypotonia, ataxia, cognitive impairment, abnormal eye movements, respiratory control disturbances and a distinctive mid-hindbrain malformation. JS demonstrates substantial phenotypic variability and genetic heterogeneity. This study provides a comprehensive view of the current genetic basis, phenotypic range and gene-phenotype associations in JS.

Mutation of ATF6 causes autosomal recessive achromatopsia.

Achromatopsia (ACHM) is an early-onset retinal dystrophy characterized by photophobia, nystagmus, color blindness and severely reduced visual acuity. Currently mutations in five genes CNGA3, CNGB3, GNAT2, PDE6C and PDE6H have been implicated in ACHM. We performed homozygosity mapping and linkage analysis in a consanguineous Pakistani ACHM family and mapped the locus to a 15.

Whole genome prediction for preimplantation genetic diagnosis.

Background: Preimplantation genetic diagnosis (PGD) enables profiling of embryos for genetic disorders prior to implantation. The majority of PGD testing is restricted in the scope of variants assayed or by the availability of extended family members. While recent advances in single cell sequencing show promise, they remain limited by bias in DNA amplification and the rapid turnaround time (<36 h) required for fresh embryo transfer.

Autosomal-Dominant Multiple Pterygium Syndrome Is Caused by Mutations in MYH3.

Multiple pterygium syndrome (MPS) is a phenotypically and genetically heterogeneous group of rare Mendelian conditions characterized by multiple pterygia, scoliosis, and congenital contractures of the limbs. MPS typically segregates as an autosomal-recessive disorder, but rare instances of autosomal-dominant transmission have been reported. Whereas several mutations causing recessive MPS have been identified, the genetic basis of dominant MPS remains unknown.

Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing.

Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly.

Haplotype-resolved genome sequencing: experimental methods and applications.

Human genomes are diploid and, for their complete description and interpretation, it is necessary not only to discover the variation they contain but also to arrange it onto chromosomal haplotypes. Although whole-genome sequencing is becoming increasingly routine, nearly all such individual genomes are mostly unresolved with respect to haplotype, particularly for rare alleles, which remain poorly resolved by inferential methods. Here, we review emerging technologies for experimentally resolving (that is, 'phasing') haplotypes across individual whole-genome sequences.

Accurate identification of centromere locations in yeast genomes using Hi-C.

Centromeres are essential for proper chromosome segregation. Despite extensive research, centromere locations in yeast genomes remain difficult to infer, and in most species they are still unknown. Recently, the chromatin conformation capture assay, Hi-C, has been re-purposed for diverse applications, including de novo genome assembly, deconvolution of metagenomic samples and inference of centromere locations.

A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis and colorectal cancer.

The genetic cause underlying the development of multiple colonic adenomas, the premalignant precursors of colorectal cancer (CRC), frequently remains unresolved in patients with adenomatous polyposis. Here we applied whole-exome sequencing to 51 individuals with multiple colonic adenomas from 48 families. In seven affected individuals from three unrelated families, we identified a homozygous germline nonsense mutation in the base-excision repair (BER) gene NTHL1.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data.

The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation.

Background: In mammals, X chromosome genes are present in one copy in males and two in females. To balance the dosage of X-linked gene expression between the sexes, one of the X chromosomes in females is silenced. X inactivation is initiated by upregulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers.

Copy-number variation and false positive prenatal aneuploidy screening results.

Investigations of noninvasive prenatal screening for aneuploidy by analysis of circulating cell-free DNA (cfDNA) have shown high sensitivity and specificity in both high-risk and low-risk cohorts. However, the overall low incidence of aneuploidy limits the positive predictive value of these tests. Currently, the causes of false positive results are poorly understood.

Massively Parallel Functional Analysis of BRCA1 RING Domain Variants.

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain.

Escape from X inactivation varies in mouse tissues.

X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi).

PI3K/AKT pathway mutations cause a spectrum of brain malformations from megalencephaly to focal cortical dysplasia.

Malformations of cortical development containing dysplastic neuronal and glial elements, including hemimegalencephaly and focal cortical dysplasia, are common causes of intractable paediatric epilepsy. In this study we performed multiplex targeted sequencing of 10 genes in the PI3K/AKT pathway on brain tissue from 33 children who underwent surgical resection of dysplastic cortex for the treatment of intractable epilepsy. Sequencing results were correlated with clinical, imaging, pathological and immunohistological phenotypes.

De novo mutations in NALCN cause a syndrome characterized by congenital contractures of the limbs and face, hypotonia, and developmental delay.

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome.

MIPSTR: a method for multiplex genotyping of germline and somatic STR variation across many individuals.

Short tandem repeats (STRs) are highly mutable genetic elements that often reside in regulatory and coding DNA. The cumulative evidence of genetic studies on individual STRs suggests that STR variation profoundly affects phenotype and contributes to trait heritability. Despite recent advances in sequencing technology, STR variation has remained largely inaccessible across many individuals compared to single nucleotide variation or copy number variation.

Exome sequencing in suspected monogenic dyslipidemias.

Background: Exome sequencing is a promising tool for gene mapping in Mendelian disorders. We used this technique in an attempt to identify novel genes underlying monogenic dyslipidemias.

Methods And Results: We performed exome sequencing on 213 selected family members from 41 kindreds with suspected Mendelian inheritance of extreme levels of low-density lipoprotein cholesterol (after candidate gene sequencing excluded known genetic causes for high low-density lipoprotein cholesterol families) or high-density lipoprotein cholesterol.

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes.

Background: The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results: We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs).

Mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia.

Focal malformations of cortical development, including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are important causes of intractable childhood epilepsy. Using targeted and exome sequencing on DNA from resected brain samples and nonbrain samples from 53 patients with FCD or HME, we identified pathogenic germline and mosaic mutations in multiple PI3K/AKT pathway genes in 9 patients, and a likely pathogenic variant in 1 additional patient. Our data confirm the association of DEPDC5 with sporadic FCD but also implicate this gene for the first time in HME.

Massively parallel single-amino-acid mutagenesis.

Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.

MAT2A mutations predispose individuals to thoracic aortic aneurysms.

Up to 20% of individuals who have thoracic aortic aneurysms or acute aortic dissections but who do not have syndromic features have a family history of thoracic aortic disease. Significant genetic heterogeneity is established for this familial condition. Whole-genome linkage analysis and exome sequencing of distant relatives from a large family with autosomal-dominant inheritance of thoracic aortic aneurysms variably associated with the bicuspid aortic valve was used for identification of additional genes predisposing individuals to this condition.

Challenges and solutions for gene identification in the presence of familial locus heterogeneity.

Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology.