Loss of LDAH associated with prostate cancer and hearing loss.

Great strides in gene discovery have been made using a multitude of methods to associate phenotypes with genetic variants, but there still remains a substantial gap between observed symptoms and identified genetic defects. Herein, we use the convergence of various genetic and genomic techniques to investigate the underpinnings of a constellation of phenotypes that include prostate cancer (PCa) and sensorineural hearing loss (SNHL) in a human subject. Through interrogation of the subject's de novo, germline, balanced chromosomal translocation, we first identify a correlation between his disorders and a poorly annotated gene known as lipid droplet associated hydrolase (LDAH). Using data repositories of both germline and somatic variants, we identify convergent genomic evidence that substantiates a correlation between loss of LDAH and PCa. This correlation is validated through both in vitro and in vivo models that show loss of LDAH results in increased risk of PCa and, to a lesser extent, SNHL. By leveraging convergent evidence in emerging genomic data, we hypothesize that loss of LDAH is involved in PCa and other phenotypes observed in support of a genotype-phenotype association in an n-of-one human subject.

De novo mutations in histone-modifying genes in congenital heart disease.

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. Here we compare the incidence of de novo mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing of parent-offspring trios. CHD cases show a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging (premature termination, frameshift, splice site) mutations. Similar odds ratios are seen across the main classes of severe CHD. We find a marked excess of de novo mutations in genes involved in the production, removal or reading of histone 3 lysine 4 (H3K4) methylation, or ubiquitination of H2BK120, which is required for H3K4 methylation. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left-right organizer. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes 'poised' promoters and enhancers, which regulate expression of key developmental genes. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

HOXA2 haploinsufficiency in dominant bilateral microtia and hearing loss.

Microtia is a rare, congenital malformation of the external ear that in some cases has a genetic etiology. We ascertained a three-generation family with bilateral microtia and hearing loss segregating as an autosomal dominant trait. Exome sequencing of affected family members detected only seven shared, rare, heterozygous, nonsynonymous variants, including one protein truncating variant, a HOXA2 nonsense change (c.703C>T, p.Q235*). The HOXA2 variant was segregated with microtia and hearing loss in the family and was not seen in 6,500 individuals sequenced by the NHLBI Exome Sequencing Project or in 218 control individuals sequenced in this study. HOXA2 has been shown to be critical for outer and middle ear development through mouse models and has previously been associated with autosomal recessive bilateral microtia. Our data extend these conclusions and define HOXA2 haploinsufficiency as the first genetic cause for autosomal-dominant nonsyndromic microtia.

ACMG clinical laboratory standards for next-generation sequencing.

Next-generation sequencing technologies have been and continue to be deployed in clinical laboratories, enabling rapid transformations in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude, and continuous advances are being made. It is now feasible to analyze an individual's near-complete exome or genome to assist in the diagnosis of a wide array of clinical scenarios. Next-generation sequencing technologies are also facilitating further advances in therapeutic decision making and disease prediction for at-risk patients. However, with rapid advances come additional challenges involving the clinical validation and use of these constantly evolving technologies and platforms in clinical laboratories. To assist clinical laboratories with the validation of next-generation sequencing methods and platforms, the ongoing monitoring of next-generation sequencing testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics has developed the following professional standards and guidelines.

Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project.

Apparently balanced chromosomal rearrangements in individuals with major congenital anomalies represent natural experiments of gene disruption and dysregulation. These individuals can be studied to identify novel genes critical in human development and to annotate further the function of known genes. Identification and characterization of these genes is the goal of the Developmental Genome Anatomy Project (DGAP). DGAP is a multidisciplinary effort that leverages the recent advances resulting from the Human Genome Project to increase our understanding of birth defects and the process of human development. Clinically significant phenotypes of individuals enrolled in DGAP are varied and, in most cases, involve multiple organ systems. Study of these individuals' chromosomal rearrangements has resulted in the mapping of 77 breakpoints from 40 chromosomal rearrangements by FISH with BACs and fosmids, array CGH, Southern-blot hybridization, MLPA, RT-PCR, and suppression PCR. Eighteen chromosomal breakpoints have been cloned and sequenced. Unsuspected genomic imbalances and cryptic rearrangements were detected, but less frequently than has been reported previously. Chromosomal rearrangements, both balanced and unbalanced, in individuals with multiple congenital anomalies continue to be a valuable resource for gene discovery and annotation.

American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants.

Genomic microarrays used to assess DNA copy number are now recommended as first-tier tests for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. Application of this technology has resulted in the discovery of widespread copy number variation in the human genome, both polymorphic variation in healthy individuals and novel pathogenic copy number imbalances. To assist clinical laboratories in the evaluation of copy number variants and to promote consistency in interpretation and reporting of genomic microarray results, the American College of Medical Genetics has developed the following professional guidelines for the interpretation and reporting of copy number variation. These guidelines apply primarily to evaluation of constitutional copy number variants detected in the postnatal setting.

Deletion of an enhancer near DLX5 and DLX6 in a family with hearing loss, craniofacial defects, and an inv(7)(q21.3q35).

Precisely regulated temporal and spatial patterns of gene expression are essential for proper human development. Cis-acting regulatory elements, some located at large distances from their corresponding genes, play a critical role in transcriptional control of key developmental genes and disruption of these regulatory elements can lead to disease. We report a three generation family with five affected members, all of whom have hearing loss, craniofacial defects, and a paracentric inversion of the long arm of chromosome 7, inv(7)(q21.3q35). High resolution mapping of the inversion showed that the 7q21.3 breakpoint is located 65 and 80 kb centromeric of DLX6 and DLX5, respectively. Further analysis revealed a 5,115 bp deletion at the 7q21.3 breakpoint. While the breakpoint does not disrupt either DLX5 or DLX6, the syndrome present in the family is similar to that observed in Dlx5 knockout mice and includes a subset of the features observed in individuals with DLX5 and DLX6 deletions, implicating dysregulation of DLX5 and DLX6 in the family's phenotype. Bioinformatic analysis indicates that the 5,115 bp deletion at the 7q21.3 breakpoint could contain regulatory elements necessary for DLX5 and DLX6 expression. Using a transgenic mouse reporter assay, we show that the deleted sequence can drive expression in the inner ear and developing bones of E12.5 embryos. Consequently, the observed familial syndrome is likely caused by dysregulation of DLX5 and/or DLX6 in specific tissues due to deletion of an enhancer and possibly separation from other regulatory elements by the chromosomal inversion.

NR2F1 deletion in a patient with a de novo paracentric inversion, inv(5)(q15q33.2), and syndromic deafness.

In an effort to discover genes important for human development, we have ascertained patients with congenital anomalies and cytogenetically balanced chromosomal rearrangements. Herein, we report a 4-year-old girl with profound deafness, a history of feeding difficulties, dysmorphism, strabismus, developmental delay, and an apparently balanced de novo paracentric chromosome 5 inversion, inv(5)(q15q33.2). Molecular cytogenetic analysis of the inversion revealed the presence of microdeletions of approximately 400-500 kb at or near both breakpoints. The 5q15 microdeletion completely removes the nuclear receptor NR2F1 (COUP-TFI) from the inverted chromosome 5. We propose haploinsufficiency of NR2F1 to be the cause of the patient's deafness and many of the other associated anomalies based on striking similarity with the Nr2f1 null mouse. Additionally, this study further highlights the need for high resolution analysis of clinical samples with chromosomal rearrangements as associated deletions may be primarily responsible for the clinical features of these patients.

X-linked deafness-2 (DFNX2) phenotype associated with a paracentric inversion upstream of POU3F4.

PURPOSE: The authors report on a 7-year-old male, designated FR, who has severe sensorineural hearing loss. Features include a round face, hypertelorism, epicanthal folds, and flat nasal root. Although there were early developmental concerns regarding FR, all but his speech delay resolved when he was placed in an educational program that accommodated his hearing loss. Genetic studies were performed to investigate genetic causes for his hearing loss. METHOD: History, physical examination, audiologic assessment, and imaging were performed according to usual practice. FMR1,GJB2,GJB6, and POU3F4 genes were sequenced. Chromosomal studies consisted of karyotyping and breakpoint analysis by fluorescence in situ hybridization (FISH). RESULTS: Results from FMR1,GJB2,GJB6, and POU3F4 sequencing and echocardiography, electrocardiogram, and abdominal ultrasound were normal. A computed tomography (CT) scan revealed a large fundus of the internal auditory canals and absence of the bony partition between the fundus and the adjacent cochlear turns, with a widened modiolus bilaterally. FR's CT findings were consistent with those described in persons with X-linked deafness-2 (DFNX2) hereditary deafness. FR's karyotype was 46,inv(X)(q13q24),Y.ish inv(X)(XIST+)mat. FISH refined the breakpoints to inv(X)(q21.1q22.3). The Xq21.1 breakpoint was narrowed to a 25-kb region 450 kb centromeric to the DFNX2 gene, POU3F4. There are rare case reports of DFNX2 patients with chromosomal rearrangements positioned centromeric to POU3F4 and no mutations within the gene. CONCLUSION: Authors hypothesized that FR's hearing loss was caused by dysregulation of POU3F4 due to separation from regulatory elements affected by the inversion.

Molecular diagnosis of hearing loss.

This unit discusses an approach to identifying a genetic etiology in an individual with nonsyndromic hearing loss. The unit begins with a discussion of the decision-making process that can be used to determine whether specific genes and/or a large gene panel should be used for molecular diagnosis of a patient presenting with nonsyndromic hearing loss. Next, two protocols are presented: (1) a full gene-sequencing assay to identify mutations in the GJB2 gene (encoding connexin 26), the most common cause of congenital hearing loss, and (2) an assay to detect the presence of the GJB6-D13S1830 deletion, a 342-kb deletion that causes hearing loss in homozygosity or in combination with a single GJB2 mutation. Finally, the unit ends with a strategy for determining the clinical significance of the test results, which can be challenging given the extensive genetic heterogeneity associated with hearing loss.