Addenda to ISCN 2020.

Since the publication of ISCN 2020, the ISCN Standing Committee have noted some clarification of the text and additional examples were needed. These addenda have already been published online (https://iscn.karger.com/) and this short report summarises the ISCN 2020 addenda for the benefit of participants. These addenda will be included in the release of the next version of ISCN.

Genome Mapping Nomenclature.

Background Genome Mapping Technologies (optical and electronic) uses ultra high-molecular weight DNA to detect structural variation and has an application in constitutional genetic disorders, haematological neoplasms and solid tumours. Genome mapping can detect balanced and unbalanced structural variation, copy number changes and haplotypes. The technique is analogous to chromosomal microarray analysis although genome mapping has the added benefit of being able to detect and ascertain the nature of more abnormalities than array, karyotyping or FISH. Key Messages This paper describes a specific nomenclature for genome mapping that can be used by diagnostic and research centres to accurately report their findings. An international nomenclature is essential for patient results to be understood by different healthcare providers as well as clear communication in publications and consistency in databases. Summary Genome mapping can detect aneuploidy, balanced and unbalanced structural variation as well as copy number changes. The Standing Committee for the International System for Human Cytogenomic Nomenclature (ISCN), recognised there was a need for a specific nomenclature for genome mapping that encompasses the range of abnormalities detected by this technique. This paper explains the general principles of the nomenclature as well as giving specific ISCN examples for the different types of numerical and structural rearrangements.

Porokeratotic eccrine ostial and dermal duct nevus associated with an 11 megabase 3p deletion.

Porokeratotic eccrine ostial and dermal duct nevus (PEODDN) is a rare eccrine hamartoma; the etiology is incompletely understood. A patient presented with congenital, widespread PEODDN. Clinical assessment, histopathologic, cytogenetic, and molecular genetic investigations on affected cells were pursued. Histopathology confirmed PEODDN, and chromosomal microarray on affected tissues identified a mosaic 3p26.3p25.3 deletion in affected tissues. This 11Mb deletion encompasses 47 OMIM genes. We propose that this and other chromosomal deletions may be implicated in some cases of PEODDN, suggesting locus heterogeneity and underscoring the importance of incorporating cytogenetic and molecular investigations into the multidisciplinary care of individuals with suspected mosaic genetic skin disorders.

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.

Atypical Hepatic Mesenchymal Hamartoma: Histologic Appearance, Immunophenotype, and Molecular Findings.

Hepatic mesenchymal hamartoma is a rare benign neoplasm principally encountered in young children. Its origin is unknown. We report an unusual hepatic mesenchymal hamartoma in a 7-month-old girl, including histopathologic findings, immunophenotype, and karyotype. Chromosomal microarray analysis of tumoral tissue and circulating lymphocytes found 4 copies of a segment at 1q44 and fluorescence in situ hybridization indicated tandem triplication, ascribed to expansion of a paternal tandem duplication. This genetic abnormality may have played a role in pathogenesis.

Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections.

A variety of laboratory methods are available for the detection of deletions of tumor suppressor genes and losses of their proteins. The clinical utility of fluorescence in situ hybridization (FISH) for the identification of deletions of tumor suppressor genes has previously been limited by difficulties in the interpretation of FISH signal patterns. The first deletion FISH assays using formalin-fixed paraffin-embedded tissue sections had to deal with a significant background level of signal losses affecting nuclei that are truncated by the cutting process of slide preparation. Recently, more efficient probe designs, incorporating probes adjacent to the tumor suppressor gene of interest, have increased the accuracy of FISH deletion assays so that true chromosomal deletions can be readily distinguished from the false signal losses caused by sectioning artifacts. This mini-review discusses the importance of recurrent tumor suppressor gene deletions in human cancer and reviews the common FISH methods being used to detect the genomic losses encountered in clinical specimens. The use of new probe designs to recognize truncation artifacts is illustrated with a four-color PTEN FISH set optimized for prostate cancer tissue sections. Data are presented to show that when section thickness is reduced, the frequency of signal truncation losses is increased. We also provide some general guidelines that will help pathologists and cytogeneticists run routine deletion FISH assays and recognize sectioning artifacts. Finally, we summarize how recently developed sequence-based approaches are being used to identify recurrent deletions using small DNA samples from tumors.

Correction to: Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections.

Figure 2 is incorrect in the original version of this article. The correct figure 2 is provided below.

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.

Variable developmental delays and characteristic facial features-A novel 7p22.3p22.2 microdeletion syndrome?

Isolated 7p22.3p22.2 deletions are rarely described with only two reports in the literature. Most other reported cases either involve a much larger region of the 7p arm or have an additional copy number variation. Here, we report five patients with overlapping microdeletions at 7p22.3p22.2. The patients presented with variable developmental delays, exhibiting relative weaknesses in expressive language skills and relative strengths in gross, and fine motor skills. The most consistent facial features seen in these patients included a broad nasal root, a prominent forehead a prominent glabella and arched eyebrows. Additional variable features amongst the patients included microcephaly, metopic ridging or craniosynostosis, cleft palate, cardiac defects, and mild hypotonia. Although the patients' deletions varied in size, there was a 0.47 Mb region of overlap which contained 7 OMIM genes: EIP3B, CHST12, LFNG, BRAT1, TTYH3, AMZ1, and GNA12. We propose that monosomy of this region represents a novel microdeletion syndrome. We recommend that individuals with 7p22.3p22.2 deletions should receive a developmental assessment and a thorough cardiac exam, with consideration of an echocardiogram, as part of their initial evaluation.

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.

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.

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.

Apparent transmission distortion of a pericentric chromosome one inversion in a large multi-generation pedigree.

Pericentric chromosome inversions are often associated with infertility, recurrent pregnancy loss, and an increased risk for offspring with congenital anomalies. We report on a chromosome 1 inversion between 1p36.21 and 1q42.13, one of the largest described familial pericentric inversions of chromosome 1. The inversion was ascertained following the birth of a female with multiple congenital anomalies due to a recombinant chromosome 1. The inversion was subsequently detected or inferred in 16 healthy individuals over five generations. Interestingly, with a ratio of 16 carriers to 6 noncarriers, there appears to be transmission distortion of the inverted chromosome 1 within the family. Although there is no reported difficulty conceiving in the family, the risk of miscarriage is higher than predicted at 34% (13/38). The recurrence risk of a recombinant chromosome also appears to be lower than expected based on the mode of ascertainment. This case contributes to the spectrum of clinical features of chromosome 1 recombinants and raises the question of whether or not there is a selective advantage of the inverted chromosome at meiosis, conception, or post-zygotically that has contributed to transmission distortion of the inverted chromosome.

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.

Discordant phenotypes in a mother and daughter with mosaic supernumerary ring chromosome 19 explained by a de novo 7q36.2 deletion and 7p22.1 duplication.

We report on a patient with severe intellectual disability, microcephaly, short stature, and dysmorphic features who, based on standard karyotyping, has two cytogenetic abnormalities: an apparently balanced paracentric inversion of chromosome 7, inv(7)(q31.2q36), and a small supernumerary ring chromosome derived entirely of material from chromosome 19. While the inversion was detected in all cells, mosaicism was observed for the ring chromosome. Interestingly, apparently identical cytogenetic abnormalities were detected in the patient's mother, who presented with normal stature, few dysmorphic features, and normal cognition without microcephaly. While the level of mosaicism could not adequately explain the phenotypic discordance, comparative genome hybridization revealed a de novo terminal deletion of chromosome 7, del(7)(q36.2), and a terminal duplication of chromosome 7, dup(7)(p22.1) in the patient. Additional cytogenetic investigation revealed that the patient inherited a recombinant chromosome derived from a cryptic maternal pericentric inversion: inv(7)(p22q36). The patient's distinctive features are consistent with the wide phenotypic spectrum reported in 7p duplication and 7q terminal deletion syndromes. These chromosomal regions contain several candidate genes of clinical significance, including SHH, EN2, and FAM20C. Our findings strongly suggest that our patient's phenotype is largely attributable to partial 7pter trisomy and partial 7qter monosomy rather than mosaic supernumerary ring chromosome 19.

HLA-DR(negative), CD34(negative) hypergranular acute myeloid leukemia with trisomy 6 and del(5)(q22q33): case report and review of the literature.

We report a unique pediatric case of hypergranular acute myeloid leukemia with myelodysplasia-related changes. The patient presented with moderate leukocytosis with neutrophilia with left-shift maturation and dysplasia, anemia, and multiple sclerotic bone lesions. The bone marrow was hypercellular with a predominance of myeloblast cells and/or abnormal promyelocytes with hypergranular cytoplasm. Flow cytometric immunophenotyping showed that the leukemic cells were positive for CD13, CD33, and myeloperoxidase, and negative for HLA-DR and CD34. Morphology and immunophenotyping were highly suggestive of acute promyelocytic leukemia. The classic t(15;17) or other RARα rearrangements were not detected by cytogenetic or molecular assays, ruling out acute promyelocytic leukemia. Standard cytogenetic analysis showed that the karyotype of the predominant clone was 47,XY,+6 with evidence of clonal evolution to 47,XY,+6,del(5)(q22q33). A literature and database review showed that trisomy 6 is a rare occurrence in hematological malignancies and, to our knowledge, has never been reported in association with del(5)(q22q33) in a child presenting with hypergranular acute myeloid leukemia with myelodysplasia-related changes. We present a current review of the literature and summarize the clinical features of 57 cases of trisomy 6 as the primary chromosomal abnormality in hematological disease.

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

OBJECTIVE: 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. METHODS: Amniotic fluid samples were prospectively categorized based on the likelihood of the fetus having a chromosome anomaly. QF-PCR results were compared with the G-banded findings. The distribution of patients and the rates of clinically significant anomalies in each risk category were determined. RESULTS: A total of 4176 amniotic fluid samples were studied. Among these, 331 cases with abnormalities were detected by both methods and an additional 19 abnormal cases were detected by G-banding only. Five of those undetected by QF-PCR were considered clinically significant, four of which were referred due to an elevated a priori risk (>4%). If QF-PCR is performed in all cases and G-banding performed only in higher risk cases, the residual risk for a clinically significant chromosome abnormality will be as low as 0.083%. CONCLUSIONS: This study suggests that QF-PCR alone is appropriate for patients with uncomplicated pregnancies, who are referred solely for an increased risk of a common trisomy.