Jean McGowan-Jordan

HGVS Recommendations for the Description of Sequence Variants: 2016 Update

The consistent and unambiguous description of sequence variants is essential to report and exchange information on the analysis of a genome. In particular, DNA diagnostics critically depends on accurate and standardized description and sharing of the variants detected. The sequence variant nomenclature system proposed in 2000 by the Human Genome Variation Society has been widely adopted and has developed into an internationally accepted standard. The recommendations are currently commissioned through a Sequence Variant Description Working Group (SVD‐WG) operating under the auspices of three international organizations: the Human Genome Variation Society (HGVS), the Human Variome Project (HVP), and the Human Genome Organization (HUGO). Requests for modifications and extensions go through the SVD‐WG following a standard procedure including a community consultation step. Version numbers are assigned to the nomenclature system to allow users to specify the version used in their variant descriptions. Here, we present the current recommendations, HGVS version 15.11, and briefly summarize the changes that were made since the 2000 publication. Most focus has been on removing inconsistencies and tightening definitions allowing automatic data processing. An extensive version of the recommendations is available online, at http://www.HGVS.org/varnomen.

De novo dup(X)(q22.3q26) in a girl with evidence that functional disomy of X material is the cause of her abnormal phenotype.

The relationship between phenotype and Xq duplications in females remains unclear. Some females are normal; some have short stature; and others have features such as microcephaly, developmental delay/mental retardation, body asymmetries, and gonadal dysgenesis. There are several hypotheses proposed in the literature to explain this variability. We describe a 7‐year‐old girl with dup(X)(q22.3q26). The pregnancy was complicated by intrauterine growth retardation, and she was distressed during labor. During her first year she fed poorly and failed to thrive. She has microcephaly, her height is at the 10th centile, and her hands and feet are strikingly small. She is hypotonic and delayed. Asymmetries of muscle power, and of leg and foot length have been noted. She has mild unilateral ptosis. She has some features of Turner syndrome, and multiple other minor anomalies such as flat labia. These are features common to other described females. This report describes our patient in detail and compares her phenotype to those of the other females with Xq duplications, displays our laboratory investigations, and discusses ideas regarding the pathogenesis of phenotype. The duplicated X is of paternal origin. It is inactivated in all cells; however, the distal duplicated portion appears to be active. We suggest that functional disomy of the duplicated X material, due to local escape from inactivation, may be responsible for the phenotype in the affected females.

The detection of chromosome anomalies by QF-PCR and residual risks as compared to G-banded analysis

To determine the detection rate of clinically significant chromosome abnormalities using quantitative fluorescent polymerase chain reaction (QF‐PCR) of fetal DNA in comparison with G‐banded analysis of cultured amniotic fluid cells and determine the residual risk if QF‐PCR were performed alone for low‐risk cases.

Nablus mask-like facial syndrome: deletion of chromosome 8q22.1 is necessary but not sufficient to cause the phenotype

Nablus mask‐like facial syndrome (NMLFS) has many distinctive phenotypic features, particularly tight glistening skin with reduced facial expression, blepharophimosis, telecanthus, bulky nasal tip, abnormal external ear architecture, upswept frontal hairline, and sparse eyebrows. Over the last few years, several individuals with NMLFS have been reported to have a microdeletion of 8q21.3q22.1, demonstrated by microarray analysis. The minimal overlapping region is 93.98–96.22 Mb (hg19). Here we present clinical and microarray data from five singletons and two mother‐child pairs who have heterozygous deletions significantly overlapping the region associated with NMLFS. Notably, while one mother and child were said to have mild tightening of facial skin, none of these individuals exhibited reduced facial expression or the classical facial phenotype of NMLFS. These findings indicate that deletion of the 8q21.3q22.1 region is necessary but not sufficient for development of the NMLFS. We discuss possible genetic mechanisms underlying the complex pattern of inheritance for this condition.

Novel clinical findings in a case of postnatally diagnosed trisomy 12 mosaicism

We report on a girl with trisomy 12 mosaicism diagnosed postnatally. She has been followed from 4 months of age for developmental delay, unilateral sensorineural hearing loss, intestinal malrotation, hemi‐hyperplasia, pigmentary dysplasia, retinopathy, and a vascular ring. To our knowledge, there have been no reports of complete trisomy 12 in the literature. However there have been a few reports describing the phenotype of individuals with trisomy 12 mosaicism. This case report is a description of the eighth liveborn individual diagnosed postnatally with this condition.

Genotype–phenotype characterization in 13 individuals with chromosome Xp11.22 duplications

We report 13 new individuals with duplications in Xp11.22‐p11.23. The index family has one male and two female members in three generations with mild‐severe intellectual disability (ID), speech delay, dysmorphic features, early puberty, constipation, and/or hand and foot abnormalities. Affected individuals were found to have two small duplications in Xp11.22 at nucleotide position (hg19) 50,112,063–50,456,458 bp (distal) and 53,160,114–53,713,154 bp (proximal). Collectively, these two regions include 14 RefSeq genes, prompting collection of a larger cohort of patients, in an attempt to delineate critical genes associated with the observed phenotype. In total, we have collected data on nine individuals with duplications overlapping the distal duplication region containing SHROOM4 and DGKK and eight individuals overlapping the proximal region including HUWE1. Duplications of HUWE1 have been previously associated with non‐syndromic ID. Our data, with previously published reports, suggest that duplications involving SHROOM4 and DGKK may represent a new syndromic X‐linked ID critical region associated with mild to severe ID, speech delay +/− dysarthria, attention deficit disorder, precocious puberty, constipation, and motor delay. We frequently observed foot abnormalities, 5th finger clinodactyly, tapering fingers, constipation, and exercise intolerance in patients with duplications of these two genes. Regarding duplications including the proximal region, our observations agree with previous studies, which have found associations with intellectual disability. In addition, expressive language delay, failure to thrive, motor delay, and 5th finger clinodactyly were also frequently observed in patients with the proximal duplication.

Severe craniosynostosis in an infant with deletion 22q11.2 syndrome

We report a male infant with 22q11.2 deletion syndrome and very severe multi‐sutural craniosynostosis associated with increased intracranial pressure, marked displacement of brain structures, and extensive erosion of the skull. While uni‐ or bi‐sultural craniosynostosis is a recognized (though relatively uncommon) feature of 22q11 deletion syndrome, a severe multi‐sutural presentation of this nature has never been reported. SNP Microarray was otherwise normal and the patient did not have common mutations in FGFR2, FGFR3, or TWIST associated with craniosynostosis. While markedly variable expressivity is an acknowledged feature of deletion 22q11 syndrome, herein we also consider and discuss the possibility that this infant may have been additionally affected with an undiagnosed single gene disorder.

Reinterpretation of sequence variants: one diagnostic laboratory’s experience, and the need for standard guidelines

PurposeThe advent of next-generation sequencing resulted in substantial increases in the number of variants detected, interpreted, and reported by molecular genetics diagnostic laboratories. Recent publications have provided standards for the interpretation of sequence variants, but there are currently no standards regarding reinterpretation of these variants. Recognizing that significant changes in variant classification may occur over time, many genetics diagnostic laboratories have independently developed practices for variant reinterpretation. The purpose of this study is to describe our laboratory approach to variant reinterpretation.MethodsWe surveyed eight genetics diagnostic laboratories in Canada and the United States.ResultsEach laboratory had differing protocols, but most felt that clinically relevant changes to variant classifications should be communicated to ordering providers. Based on results of this survey and our experience, we developed a cost-effective and resource-efficient approach to variant reinterpretation.ConclusionOngoing variant reinterpretation is required to maintain the highest standards for delivering genetics laboratory services. Our approach to variant reinterpretation offers an efficient solution that does not compromise accuracy or timely delivery of genetics laboratory services.

Abstract 63: Incorporation of flanking probes reduces truncation losses for fluorescence in situ hybridization analysis of recurrent genomic deletions in tumor sections.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Fluorescence in situ hybridization (FISH) is a robust technique when used with appropriate interpretative guidelines, and the assay can yield consistent results that provide diagnostically and prognostically useful information to help guide patient therapy. The establishment of quality control standards in clinical laboratories using routine FISH analysis of formalin fixed paraffin embedded (FFPE) sections in different tumor types for fusion and break-apart strategies and gene amplifications have been developed for both analysis and reporting, but at the present time there are no comprehensive guidelines for the analysis of genomic deletions using FFPE sections. The use of FISH on archival FFPE samples is technically demanding and becomes more challenging when applied to paraffin-embedded tissue microarrays. The evaluation of FISH signals in interphase nuclei of FFPE sections is affected by truncation and overlapping of the nuclei due to varying cell density, tissue architecture differences and histological forms. In this study we report a generalizable four-color deletion FISH approach to assist interpretation problems arising when evaluating FISH signals. The guidelines will help address interpretative dilemmas associated with overlapping and truncated nuclei in FFPE prostate cancer sections. The four-color FISH approach was developed using the PTEN tumor suppressor gene deletion model in prostate cancer. The PTEN assay was based on a robust bioinformatics analysis of 311 published human genome array datasets and comprises a centromeric “chromosome enumeration” probe, a specific PTEN gene probe, and control flanking probes either side of the target probe. The sensitivity and specificity parameters of the four-color PTEN probe set were further characterized using a large number of well-characterized tumors and stringent scoring criteria. The incorporation of flanking control probes allowed the analysts to determine if the chromosomal region was subject to truncation loss. A minimum threshold for apparent deletion frequency was set to address the heterogeneous and homogeneous nature of tumor histology. In addition the approach facilitated analysis of genotypic heterogeneity and varying clonality within different foci of tumor in the prostate. Overall the approach provided robust and highly reproducible results that minimized inter- and intra-assay variability. The four-color FISH deletion assay reduced the frequency of misinterpretation and improved both the quality and throughput of FISH analyses using clinical samples. Moreover the use of established controls and conservative cut-offs for assigning deletions will facilitate more coherent approach to developing reporting standards for deletion assays as more tumor suppressor genes of clinical importance are discovered by next generation sequencing methods. Citation Format: Maisa Yoshimoto, Olga Ludkovski, Jennifer Good, Robert J. Gooding, Jean McGowan-Jordan, Alexander Boag, Andrew Evans, Ming-Sound Tsao, Paulo Nuin, Jeremy A. Squire. Incorporation of flanking probes reduces truncation losses for fluorescence in situ hybridization analysis of recurrent genomic deletions in tumor sections. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 63. doi:10.1158/1538-7445.AM2013-63

Leveraging the power of new molecular technologies in the clinical setting requires unprecedented awareness of limitations and drawbacks: experience of one diagnostic laboratory

Background Advances in molecular technologies and in-silico variant prediction tools offer wide-ranging opportunities in diagnostic settings, yet they also present with significant limitations. Objective Here, we contextualise the limitations of next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA) and in-silico prediction tools routinely used by diagnostic laboratories by reviewing specific experiences from our diagnostic laboratory. Methods We investigated discordant annotations and/or incorrect variant ‘callings’ in exons of 56 genes constituting our cardiomyopathy and connective tissue disorder NGS panels. Discordant variants and segmental duplications (SD) were queried using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool and the University of California Santa Cruz genome browser, respectively, to identify regions of high homology. Discrepant variant analyses by in-silico models were re-evaluated using updated file entries. Results We observed a 5% error rate in MYH7 variant ‘calling’ using MLPA, which resulted from >90% homology of the MYH7 probe-binding site to MYH6. SDs were detected in TTN, PKP2 and MYLK. SDs in MYLK presented the highest risk (15.7%) of incorrect variant ‘calling’. The inaccurate ‘callings’ and discrepant in-silico predictions were resolved following detailed investigation into the source of error. Conclusion Recognising the limitations described here may help avoid incorrect diagnoses and leverage the power of new molecular technologies in diagnostic settings.