Limb Malformations Panel

Summary
  • Is a 45 gene panel that includes assessment of non-coding variants
  • Is ideal for patients with congenital limb reduction defects or split hand / foot anomalies that can be isolated or syndromic.

Analysis methods
  • PLUS
  • SEQ
  • DEL/DUP
Availability

4 weeks

Number of genes

45

Test code

MA4001

Panel size

Large

CPT codes
SEQ 81479
DEL/DUP 81479

Summary

The Blueprint Genetics Limb Malformations Panel (test code MA4001):

ICD codes

Commonly used ICD-10 code(s) when ordering the Limb Malformations Panel

ICD-10 Disease
Q87.1 Cornelia de Lange syndrome
Q87.2 Holt-Oram syndrome
Q87.2 Adams-Oliver syndrome
D61.09 Fanconi anemia

Sample Requirements

  • Blood (min. 1ml) in an EDTA tube
  • Extracted DNA, min. 2 μg in TE buffer or equivalent
  • Saliva (Oragene DNA OG-500 kit/OGD-500 or OG-575 & OGD-575)

Label the sample tube with your patient's name, date of birth and the date of sample collection.

Note that we do not accept DNA samples isolated from formalin-fixed paraffin-embedded (FFPE) tissue. Read more about our sample requirements here.

Limb malformations are present in isolated limb reduction defects and split hand/foot anomalies as well as some syndromic forms, such as the Cornelia de Lange syndrome caused by mutations in the NIPBL, RAD21, SMC3, HDAC8 or SMC1A genes, the Adams-Oliver syndrome caused by the ARHGAP31, DLL4, DOCK6, EOGT, NOTCH1 or RBPJ genes and the Fanconi-Anemia syndrome caused most commonly by the FANCA, FANCC or FANCG genes. Limb reduction defects are congenital limb anomalies that can affect thumb or radius of the hand (thumb/radial hypoplasia or aplasia) or cause transverse terminal or longitudinal reduction defects or hypoplasia of the limb. These limb anomalies can be unilateral or bilateral and only hands, only feet or all limbs can be affected. Different genetic and also non-genetic etiologies can cause very similar limb phenotypes sometimes making the clinical diagnostics of these anomalies challenging. Mutations of TBX5 cause Holt-Oram syndrome, characterized by a combination of cardiac defects and upper limb malformations. Thrombocytopenia-Absent Radius (TAR) syndrome is caused by mutations in RBM8A gene and is characterized by bilateral absence of the radii and thrombocytopenia, thumbs are usually present. SALL4 mutations can cause Duane Radial Ray (Okihiro) syndrome with varying degree of radial ray hypoplasia and Duane anomaly. Split hand/foot malformation (SHFM) refers to a rare congenital malformation with median clefts of the hands and feet. SHFM can be isolated or syndromic is genetically heterozygous. The most typical form of inheritance is autosomal dominant with incomplete penetrance.

Genes in the Limb Malformations Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
ARHGAP31 Adams-Oliver syndrome AD 3 6
ATR Cutaneous telangiectasia and cancer syndrome, Seckel syndrome AD/AR 10 33
BHLHA9 Syndactyly Malik-Percin type, mesoaxial synostotic, with phalangeal reduction, Split hand-foot malformation with long bone deficiency (SHFLD3), Gollop-Wolfgang AR 4 43
BRCA2 Fanconi anemia, Medulloblastoma, Glioma susceptibility, Pancreatic cancer, Wilms tumor, Breast-ovarian cancer, familial AD/AR 3369 2659
BRIP1 Fanconi anemia, Breast cancer AD/AR 238 189
DHODH Postaxial acrofacial dysostosis (Miller syndrome) AR 8 20
DLL4 Adams-Oliver syndrome AD 13 14
DLX5 Split-hand/foot malformation with sensorineural hearing loss AR 3 9
DOCK6 Adams-Oliver syndrome AR 21 21
EOGT Adams-Oliver syndrome AR 8 5
ERCC4 Fanconi anemia, Xeroderma pigmentosum, XFE progeroid syndrome AR 13 70
ESCO2 SC phocomelia syndrome, Roberts syndrome AR 30 31
FANCA Fanconi anemia AR 191 677
FANCB Fanconi anemia XL 11 21
FANCC Fanconi anemia AR 94 64
FANCD2* Fanconi anemia AR 21 61
FANCE Fanconi anemia AR 4 17
FANCF Fanconia anemia AR 7 16
FANCG Fanconi anemia AR 16 92
FANCI Fanconi anemia AR 13 45
FANCL Fanconi anemia AR 13 24
FANCM Fanconi anemia AR 6 50
FGF10 Aplasia of lacrimal and salivary glands AD 15 13
GDF5 Multiple synostoses syndrome, Fibular hypoplasia and complex brachydactyly, Acromesomelic dysplasia, Hunter-Thompson, Symphalangism, proximal, Chondrodysplasia, Brachydactyly type A2, Brachydactyly type C, Grebe dysplasia AD/AR 23 53
HDAC8 Cornelia de Lange syndrome XL 41 50
NIPBL Cornelia de Lange syndrome AD 311 425
NOTCH1 Aortic valve disease AD 56 96
NSDHL Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD syndrome), CK syndrome XL 15 28
PALB2 Fanconi anemia, Pancreatic cancer, Breast cancer AD/AR 495 406
RAD21* Cornelia de Lange syndrome 4 AD 14 11
RAD51C Fanconi anemia, Breast-ovarian cancer, familial AD/AR 107 125
RBM8A*,# Thrombocytopenia - absent radius AD/AR 5 12
RBPJ* Adams-Oliver syndrome AD 7 6
RECQL4 Baller-Gerold syndrome, RAPADILINO syndrome, Rothmund-Thomson syndrome AR 82 114
SALL1* Townes-Brocks syndrome 1 AD 31 87
SALL4 Acro-renal-ocular syndrome, Duane-radial ray/Okohiro syndrome AD 21 56
SF3B4 Acrofacial dysostosis 1, Nager AD 27 38
SLX4 Fanconi anemia AR 18 72
SMC1A Cornelia de Lange syndrome XL 73 87
SMC3 Cornelia de Lange syndrome AD 25 21
TBX3 Ulnar-Mammary syndrome AD 6 20
TBX5 Holt-Oram syndrome AD 61 127
TP63 Rapp-Hodgkin syndrome, Orofacial cleft, ADULT syndrome, Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome, Ankyloblepharon-ectodermal defects-cleft lip/palate, Split-hand/foot malformation, Limb-mammary syndrome AD 59 122
WNT7A Ulna and fibula, absence of, with severe limb deficiency (Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome), Fuhrmann syndrome AR 6 11
XRCC2 Hereditary breast cancer AD/AR 10 21

* Some, or all, of the gene is duplicated in the genome. Read more.

# The gene has suboptimal coverage (means <90% of the gene’s target nucleotides are covered at >20x with mapping quality score (MQ>20) reads).

The sensitivity to detect variants may be limited in genes marked with an asterisk (*) or number sign (#)

Gene refers to the HGNC approved gene symbol; Inheritance refers to inheritance patterns such as autosomal dominant (AD), autosomal recessive (AR), X-linked (XL), X-linked dominant (XLD) and X-linked recessive (XLR); ClinVar refers to the number of variants in the gene classified as pathogenic or likely pathogenic in this database (ClinVar); HGMD refers to the number of variants with possible disease association in the gene listed in Human Gene Mutation Database (HGMD). The list of associated, gene specific phenotypes are generated from CGD or Orphanet databases.

Non-coding variants covered by Limb Malformations Panel

Gene Genomic location HG19 HGVS RefSeq RS-number
BRCA2 Chr13:32889805 c.-40+1G>A NM_000059.3
BRCA2 Chr13:32953872 c.8954-15T>G NM_000059.3
BRCA2 Chr13:32971007 c.9502-28A>G NM_000059.3 rs397508059
BRIP1 Chr17:59858864 c.1629-498A>T NM_032043.2
FANCA Chr16:89816056 c.3239+82T>G NM_000135.2
FANCA Chr16:89818822 c.2982-192A>G NM_000135.2
FANCA Chr16:89831215 c.2778+83C>G NM_000135.2 rs750997715
FANCA Chr16:89836111 c.2504+134A>G NM_000135.2
FANCA Chr16:89836805 c.2223-138A>G NM_000135.2
FANCA Chr16:89849346 c.1567-20A>G NM_000135.2 rs775154397
FANCA Chr16:89864654 c.893+920C>A NM_000135.2
FANCD2 Chr3:10083186 c.696-121C>G NM_033084.3
FANCI Chr15:89825208 c.1583+142C>T NM_001113378.1
NIPBL Chr5:36877266 c.-94C>T NM_133433.3
NIPBL Chr5:36953718 c.-79-2A>G NM_133433.3
NIPBL Chr5:37022138 c.5329-15A>G NM_133433.3 rs587783968
PALB2 Chr16:23649285 c.109-12T>A NM_024675.3 rs774949203
TBX5 Chr12:114704515 c.*88822C>A NM_000192.3 rs141875471

Test Strengths

The strengths of this test include:
  • CAP and ISO-15189 accreditations covering all operations at Blueprint Genetics including all Whole Exome Sequencing, NGS panels and confirmatory testing
  • CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory
  • Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance
  • Careful construction of clinically effective and scientifically justified gene panels
  • Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level
  • Our publically available analytic validation demonstrating complete details of test performance
  • ~1,500 non-coding disease causing variants in Blueprint WES assay (please see below ‘Non-coding disease causing variants covered by this panel’)
  • Our rigorous variant classification based on modified ACMG variant classification scheme
  • Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data
  • Our comprehensive clinical statements

Test Limitations

The following exons are not included in the panel as they are not sufficiently covered with high quality sequence reads: RBM8A (3). Genes with suboptimal coverage in our assay are marked with number sign (#) and genes with partial, or whole gene, segmental duplications in the human genome are marked with an asterisk (*) if they overlap with the UCSC pseudogene regions. Gene is considered to have suboptimal coverage when >90% of the gene's target nucleotides are not covered at >20x with mapping quality score (MQ>20) reads. The technology may have limited sensitivity to detect variants in genes marked with these symbols (please see the Panel content table above).

This test does not detect the following:
  • Complex inversions
  • Gene conversions
  • Balanced translocations
  • Mitochondrial DNA variants
  • Repeat expansion disorders unless specifically mentioned
  • Non-coding variants deeper than ±20 base pairs from exon-intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).
This test may not reliably detect the following:
  • Low level mosaicism (variant with a minor allele fraction of 14.6% is detected with 90% probability)
  • Stretches of mononucleotide repeats
  • Indels larger than 50bp
  • Single exon deletions or duplications
  • Variants within pseudogene regions/duplicated segments

The sensitivity of this test may be reduced if DNA is extracted by a laboratory other than Blueprint Genetics.

For additional information, please refer to the Test performance section and see our Analytic Validation.

The Blueprint Genetics limb malformations panel covers classical genes associated with Cornelia de Lange syndrome, Holt-Oram syndrome, Adams-Oliver syndrome, Fanconi anemia, split-hand/foot malformation, Duane-radial ray/Okohiro syndrome and Townes-Brocks syndrome 1. The genes on the panel have been carefully selected based on scientific literature, mutation databases and our experience.

Our panels are sliced from our high-quality whole exome sequencing data. Please see our sequencing and detection performance table for different types of alterations at the whole exome level (Table).

Assays have been validated for different starting materials including EDTA-blood, isolated DNA (no FFPE), saliva and dry blood spots (filter card) and all provide high-quality results. The diagnostic yield varies substantially depending on the assay used, referring healthcare professional, hospital and country. Blueprint Genetics' Plus Analysis (Seq+Del/Dup) maximizes the chance to find a molecular genetic diagnosis for your patient although Sequence Analysis or Del/Dup Analysis may be a cost-effective first line test if your patient's phenotype is suggestive of a specific mutation type.

Performance of Blueprint Genetics Whole Exome Sequencing (WES) assay. All individual panels are sliced from WES data.

Sensitivity % (TP/(TP+FN) Specificity %
Single nucleotide variants 99.65% (412,456/413,893) >99.99%
Insertions, deletions and indels by sequence analysis
1-10 bps 96.94% (17,070/17,608) >99.99%
11-50 bps 99.07% (957/966) >99.99%
Copy number variants (exon level dels/dups)
Clinical samples (small CNVs, n=52)
1 exon level deletion 92.3% (24/26) NA
2 exons level deletion/duplication 100.0% (11/11) NA
3-7 exons level deletion/duplication 93.3% (14/15) NA
Microdeletion/-duplication sdrs (large CNVs, n=37))
Size range (0.1-47 Mb) 100% (37/37)
Simulated CNV detection
2 exons level deletion/duplication 90.98% (7,357/8,086) 99.96%
5 exons level deletion/duplication 98.63% (7,975/8,086) 99.98%
     
The performance presented above reached by WES with the following coverage metrics
     
Mean sequencing depth at exome level 174x
Nucleotides with >20x sequencing coverage (%) 99.4%

Bioinformatics

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases such as, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as SIFT, PolyPhen, MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, the customer has an access to details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with inadequate coverage if present. This reflects our mission to build fully transparent diagnostics where customers have easy access to crucial details of the analysis process.

Clinical interpretation

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the corner stone of clinical interpretation and resulting patient management decisions. Our classifications follow the Blueprint Genetics Variant Classification Schemes based on the ACMG guideline 2015. Minor modifications were made to increase reproducibility of the variant classification and improve the clinical validity of the report. Our experience with tens of thousands of clinical cases analyzed at our laboratory allowed us to further develop the industry standard.

The final step in the analysis is orthogonal confirmation. Sequence variants classified as pathogenic, likely pathogenic and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing when they do not meet our stringent NGS quality metrics for a true positive call.
Reported copy number variations with a size <10 exons are confirmed by orthogonal methods such as qPCR if the specific CNV has been seen less than three times at Blueprint Genetics (Plus analysis only).

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts and detailed information about related phenotypes. We also provide links to the references used, congress abstracts and mutation databases to help our customers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification within the family. In the case of variants of uncertain significance (VUS), we do not recommend family member risk stratification based on the VUS result. Furthermore, in the case of VUS, we do not recommend the use of genetic information in patient management or genetic counseling.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Thus, our database, and our understanding of variants and related phenotypes, is growing by leaps and bounds. Our laboratory is therefore well positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering health care provider at no additional cost.

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