This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.

The great majority of tests are completed within 20 days.

Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) and genitopatellar syndrome (GPS) are rare disorders with several overlapping clinical features. Symptoms common to both syndromes include developmental delay; congenital hearts defects; hearing loss; skeletal deformities; and dental, thyroid, and genital anomalies (Campeau et al. 2012). SBBYSS can be distinguished by characteristic dysmorphic features that include blepharophimosis, bulbous nasal tip, small mouth, a thin upper lip and immobile mask-like face; long thumbs and great toes; and lacrimal duct anomalies. Genital anomaly, in the form of cryptorchidism, is present in males only. Symptoms at onset include hypotonia and feeding difficulties; and appear usually in infancy (Ohdo et al. 1986; Say and Barber 1987; Biesecker, 1991; Clayton-Smith et al. 2011). The hallmark features of GPS include hypoplasia or agenesis of the patellae; flexion deformities of the hips and knees and club feet; kidneys anomalies in the form of renal cysts and hydronephrosis; and agenesis of the corpus callosum with microcephaly. Genital anomalies are present in both males and females. Additional features include facial dysmorphism; brachydactyly; and pulmonary hypoplasia. Symptoms begin usually in infancy, although prenatal onset has been reported (Cormier-Daire et al. 2000; Reardon et al. 2002; Penttinen et al. 2009) Noonan Syndrome (NS) is characterized by dysmorphic facial features, growth and congenital heart defects, and musculoskeletal abnormalities. Cardiac abnormalities are found in up to 80% of patients and include pulmonary valve stenosis, atrial septal defect, atrioventricular canal defect, and hypertrophic cardiomyopathy. Musculoskeletal abnormalities include short stature, chest deformity with sunken or raised sternum, and short webbed neck. Several additional abnormalities have been described and include renal, genital, hematological, neurologic, cognitive, behavioral, gastrointestinal, dental, and lymphatic findings. Intelligence is usually normal; however, learning disabilities may be present. NS is characterized by extensive clinical heterogeneity, even among members of the same family. Diagnosis is often made in infancy or early childhood. Symptoms often change and lessen with advancing age. The prevalence of NS is estimated at 1 in 1000 to 1 in 2,500 births worldwide (Allanson et al. 1985; Romano et al. 2010; Smpokou et al. 2012).

Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) and genitopatellar syndrome (GPS) are both caused by heterozygous pathogenic variants in the KAT6B gene (Clayton-Smith et al. 2011; Campeau et al AJHG. 2012; Simpson et al. 2012; Soden et al. 2014). Variants are all truncating and include nonsense, small deletions and insertions, and indels. All variants occurred de novo. About 10 GPS-causative variants have been reported to date. They are all clustered in the proximal part of the last exon and lead to a truncated protein lacking the activating domain. Twelve variants were reported in patients with SBBYSS. The majority are distributed throughout the entire length of the gene and lead to nonsense-mediated decay. A few variants occured more distally in the last exon (Campeau et al. 2012). Defects in the KAT6B gene appear to be a rare cause of Noonan syndrome. To date, only one patient with a Noonan-like syndrome phenotype was reported to have a de novo balanced translocation in KAT6B. Clinical features in the latter patient overlap with those of Noonan syndrome and included short stature; distinct facial features with blepharophimosis, ptosis, high arched eyebrows, low-set ears with overfolded helix and fleshy lobe, smooth philtrum, retrognathia, and a high arched palate; and attention deficit, hyperactivity disorder, and learning disability (Kraft et al. 2011). At least 70% of patients with a diagnosis of Noonan syndrome have heterozygous germline pathogenic variants in twelve genes (Romano et al. 2010; Tartaglia et al. 2010; Aoki et al. 2013). Ten of these genes (PTPN11, SOS1, RAF1, KRAS, HRAS, SHOC2, BRAF, NRAS, MAP2K1 and MAP2K1) encode components of the main Ras/MAPK signaling pathway; while CBL and RIT1 encode proteins that are involved in its regulation (Rauen 2013; Martinelli et al. 2010; Niemeyer et al. 2010; Kraft et al. 2011; Aoki et al. 2013). The KAT6B gene, also known as MYST4, encodes one of five members of the MYST histone acetyltransferase (MYST HAT) family that is involved in histone acetylation, which is a characteristic of actively transcribed genes (MacDonald et al. 2009).

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This test provides full coverage of all coding exons of the KAT6B gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Patients with clinical features of SBBYSS or GPS and their family members are candidates for this test. Patients with clinical features suggestive of Noonan syndrome and no pathogenic variants in the PTPN11, SOS1, RAF1, KRAS, HRAS, SHOC2, BRAF, NRAS, MAP2K1, MAP2K1,CBL or RIT1 genes are also candidates for this test.

Diseases

NextGen Sequencing

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Deletion and Duplication Testing via NGS

NextGen Sequencing

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

NextGen Sequencing

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~10 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.