Panels, Exomes, Genomes, and More-Finding the Best Path Through the Diagnostic Odyssey.

Selecting the ideal test to evaluate an individual with a suspected genetic disorder can be challenging. While several clinical testing options are available, no single test yet captures all potentially causative genetic variants. Thus, clinicians may order testing in a stepwise fashion, and what to order after non-diagnostic testing can be challenging to determine. Here, we provide an overview of commonly used clinical genetic tests, guidance on when they are best used, and what they may miss. We conclude with a discussion of how new technologies might be used to identify challenging variants and simplify clinical testing in the future.

ANKLE2-related microcephaly: A variable microcephaly syndrome resembling Zika infection.

OBJECTIVE: This study delineates the clinical and molecular spectrum of ANKLE2-related microcephaly (MIC), as well as highlights shared pathological mechanisms between ANKLE2 and the Zika virus. METHODS: We identified 12 individuals with MIC and variants in ANKLE2 with a broad range of features. Probands underwent thorough phenotypic evaluations, developmental assessments, and anthropometric measurements. Brain imaging studies were systematically reviewed for developmental abnormalities. We functionally interrogated a subset of identified ANKLE2 variants in Drosophila melanogaster. RESULTS: All individuals had MIC (z-score ≤ -3), including nine with congenital MIC. We identified a broad range of brain abnormalities including simplified cortical gyral pattern, full or partial callosal agenesis, increased extra-axial spaces, hypomyelination, cerebellar vermis hypoplasia, and enlarged cisterna magna. All probands had developmental delays in at least one domain, with speech and language delays being the most common. Six probands had skin findings characteristic of ANKLE2 including hyper- and hypopigmented macules. Only one individual had scalp rugae. Functional characterization in Drosophila recapitulated the human MIC phenotype. Of the four variants tested, p.Val229Gly, p.Arg236*, and p.Arg536Cys acted as partial-loss-of-function variants, whereas the c.1421-1G>C splicing variant demonstrated a strong loss-of-function effect. INTERPRETATION: Deleterious variants in the ANKLE2 gene cause a unique MIC syndrome characterized by congenital or postnatal MIC, a broad range of structural brain abnormalities, and skin pigmentary changes. Thorough functional characterization has identified shared pathogenic mechanisms between ANKLE2-related MIC and congenital Zika virus infection. This study further highlights the importance of a thorough diagnostic evaluation including molecular diagnostic testing in individuals with MIC.

Targeted long-read sequencing identifies missing disease-causing variation.

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.

Congenital virilization of female infants recognized after pregnancies with retained levonorgestrel intrauterine devices.

The Mirena intrauterine device (IUD) is a hormone-secreting contraceptive device. Pregnancy with the Mirena is rare and effects to the fetus are unknown. Here we present four females with genital virilization after pregnancy with persistent Mirena IUD. All patients had a 46, XX karyotype and normal hormone evaluation. All underwent exam under anesthesia, demonstrating posterior labial fusion and short urogenital sinus with normal bladder, urethra, vagina, and cervix. Three of four patients underwent flap vaginoplasty without complications and good cosmetic outcomes. This series suggests that persistent levonorgestrel-secreting IUD during pregnancy is associated with genital virilization in female fetuses.

A child with autism, behavioral issues, and dysmorphic features found to have a tandem duplication within CTNND2 by mate-pair sequencing.

We describe a 5-year-old male with developmental delay, behavioral problems, and dysmorphic features who was found by microarray to have a 93-kb duplication of uncertain significance that fully encompasses the third exon of CTNND2 (delta catenin). Mate-pair sequencing was used to determine that the duplication is tandem and is predicted to lead to CTNND2 haploinsufficiency. Haploinsufficiency for CTNND2 has been shown to result in developmental delay and intellectual disability, providing a unifying diagnosis for this patient. His features overlap those associated with the larger cri-du-chat deletion of this region, expanding the clinical phenotype of isolated CTNND2 variants. The use of mate-pair sequencing to determine the orientation of the small duplication was essential to the diagnosis and avoided the use of exome sequencing, which would not have defined the arrangement of the duplication. This is only the second reported patient, to our knowledge, with a single exon duplication of CTNND2.