Hypophosphatemic Nephrolithiasis/Osteoporosis-2 (NPHLOP2) via the SLC9A3R1 Gene
- Summary and Pricing
- Clinical Features and Genetics
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The great majority of tests are completed within 18 days.
In the original report of SLC9A3R1-associated nephrolithiasis, 4 of 92 (4.3%) unrelated patients with either nephrolithiasis or bone demineralization were found to have a heterozygous pathogenic SLC9A3R1 variant (Karim et al. 2008).
In an international cohort comprising 272 genetically unresolved individuals (106 children and 166 adults) from 268 families with nephrolithiasis (n=256) or isolated nephrocalcinosis (n=16), Halbritter et al. detected likely (or suspected) causative variants in 14 of 30 analyzed genes, leading to a molecular diagnosis in 14.9% (40 of 268) of all cases (Halbritter et al. 2015). Two families (0.75%) were found to have a heterozygous pathogenic SLC9A3R1 variant.
In another international pediatric cohort comprising 143 individuals under 18 years of age, with nephrolithiasis (n=123) or isolated nephrocalcinosis (n=20), Braun et al. detected likely (or suspected) causative variants in 14 of the same set of 30 genes known nephrolithiasis/nephrocalcinosis genes, leading to a molecular diagnosis in 16.8% (24 of 143) (Braun et al. 2016). One individual (0.7%) was found to have a heterozygous pathogenic SLC9A3R1 variant.
Nephrolithiasis is a clinical condition in which urinary supersaturation leads to stone formation in the urinary system (the lumen of the collecting system, ureter, and bladder) (Braun et al. 2016; Halbritter et al. 2015; Gee et al. 2016). Patients with kidney stones may have no symptoms or may present with flank pain, gross or microscopic hematuria, obstruction of one or both kidneys, and urinary infections. It affects 10%-15% of adults in their lifetime. SLC9A3R1-associated nephrolithiasis presents with hypophosphatemia and decreased renal phosphate resorption characterized by significantly decreased tubular maximum for phosphate resorption per glomerular filtration rate (TmP/GFR) (Karim et al. 2008). Some patients may also have a spinal deformity and decreased bone mineral density (BMD).
SLC9A3R1-associated nephrolithiasis is an autosomal dominant disorder (Karim et al. 2008; Halbritter et al. 2015). The SLC9A3R1 gene (six coding exons) encodes sodium-hydrogen exchanger regulatory factor 1 (NHERF1), which controls renal phosphate transport. To date, documented genetic defects of SLC9A3R1 include both missense and truncating variants (Human Gene Mutation Database). Large deletions/duplications have not been reported to date.
This test involves bidirectional Sanger DNA sequencing of all coding exons of the SLC9A3R1 gene. The entire coding region and ~20 bp of flanking non-coding DNA on either side of each splice site are sequenced. We will also sequence any single exon (Test #100) in family members of patients with a known pathogenic variant or to confirm research results.
Indications for Test
Candidates for this test are patients with hypophosphatemic nephrolithiasis/osteoporosis-2 (NPHLOP2). Testing is also indicated for family members of patients who have known pathogenic variants in the SLC9A3R1 gene.
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|Nephrolithiasis and Nephrocalcinosis Sequencing Panel|
- Genetic Counselor Team - email@example.com
- Wuyan Chen, PhD - firstname.lastname@example.org
- Braun D.A. et al. 2016. Clinical Journal of the American Society of Nephrology. 11: 664-72. PubMed ID: 26787776
- Gee H.Y. et al. 2016. American Journal of Human Genetics. 98: 1228-34. PubMed ID: 27210743
- Halbritter J. et al. 2015. Journal of the American Society of Nephrology. 26: 543-51. PubMed ID: 25296721
- Human Gene Mutation Database (Bio-base).
- Karim Z. et al. 2008. The New England Journal of Medicine. 359: 1128-35. PubMed ID: 18784102
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all 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 for example 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 and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.