# Presentation: 008367

## Biochemical & MolecularCollaborationsProband / FamilyTest Examples

** **

- Andy Faucett, MS, CGC
- Emory University School of Medicine

*Note: All information in this presentation is from GeneReviews.*
*Content was condensed for slide format.*

## Tay-Sachs Testing - Proband

**Tay-Sachs = Hexosaminidase A Deficiency**
**Assay of HEX A enzymatic activity is the primary method of diagnosis for symptomatic individuals and of population screening ... given its greater sensitivity.**
**Issue**
**Borderline activity levels**

## Tay-Sachs Testing - Proband

**Targeted mutation analysis of common *****HEXA mutations***** can be used for: **
**Symptomatic individual to identify the disease-causing mutations;**
_**Confirmatory diagnosis**_** in symptomatic individuals with **_**borderline **_**enzyme activity. **
**Sequence analysis or mutation scanning would be required in an individual who: **
**Is affected but had only one or neither mutations identified using a panel of standard mutations.**

## Tay-Sachs Testing - Family

**Assay of HEX A enzymatic activity using **_**synthetic substrates**_** provides a simple, inexpensive, and highly accurate method for carrier identification.**
**Serum may be used to test **_**all males**_** and those **_**women who are not pregnant and not using oral contraceptives; **_
_**Leukocytes**_** are used to test: (1) pregnant women; (2) women using oral contraceptives; and (3) any individual whose serum HEX A enzymatic activity is in an inconclusive range. **
**Issue – pseudodeficiency allele**

## Pseudodeficiency AlleleNeed for Molecular Clarification

**One pseudodeficiency allele reduces HEX A enzymatic activity toward **_**synthetic substrates**_**, but does not with the **_**natural substrate**_
**All enzymatic assays use the artificial substrate - the naturally occurring GM2 ganglioside is not stable and not available.**
**Molecular genetic testing can be used to clarify.**
**About 35% of non-Jewish individuals identified as carriers by HEX A enzyme-based testing are carriers of a pseudodeficiency allele. **
**About 2% of Jewish individuals identified as carriers by HEX A enzyme-based testing are actually heterozygous for a pseudodeficiency allele.**

## Tay-Sachs Testing - Family

**Mutation analysis with a panel of common *****HEXA***** mutations: **
**Screen Ashkenazi Jewish individuals for the three common mutations which account for 92% to 94% of carriers.**
**Distinguish pseudodeficiency from disease-causing alleles.**
**Identify the specific disease-causing alleles in an affected individual for carrier detection in **_**at-risk family members. **_
**Prenatal diagnosis when both parental mutations are known. **
**Sequence analysis or mutation scanning would be required to identify *****HEXA***** mutations in an individual who:  **
**Has carrier-level enzymatic results but did not have a mutation identified using a panel of standard mutations. **

## TS Molecular Test Methods

**Targeted mutation analysis.  Panel of the six most common mutations: **
**Three null alleles, (+TATC1278, +1IVC12, +1IVS9), which are associated with TSD in the homozygous state or as compound heterozygotes.**
**G269S allele, which is associated with the adult-onset form of hexosaminidase A deficiency in the homozygous state or in compound heterozygosity with a null allele. **
**Two pseudodeficiency alleles (R247W and R249W), which are not associated with neurologic disease but with reduced degradation of synthetic substrate.**
**Extended panels or testing for selected mutations specific to certain populations. In Quebec, a 7.6-kb deletion is the most common. **
**In the French-Canadian population or other populations with founder mutations, testing for the appropriate mutations is needed.**
**Sequence analysis/mutation scanning.  100+ *****HEXA***** mutations have been detected to date.**

## TS Mutation Detection

## Mucopolysaccharidoses Type 1Proband

**Hurler, Hurler-Scheie, and Scheie Syndrome**
**Diagnosis/testing.  The diagnosis of MPS I relies on the demonstration of deficient activity of the lysosomal enzyme  -L-iduronidase in peripheral blood leukocytes or cultured fibroblasts. **
**Glycosaminoglycan (GAG) (heparan and dermatan sulphate) urinary excretion is a useful preliminary test.**

## MPS 1 – Diagnostic Testing

***IDUA***** is the only **_**[gene](http://www.genetests.org/servlet/access?qry=66&db=genestar&fcn=term&gtreport2=true&id=8888891&key=1BAn8AY2sg49i&format=frame)**__** currently known to be associated with MPS I. Using **__**[sequence analysis](http://www.genetests.org/servlet/access?qry=198&db=genestar&fcn=term&gtreport2=true&id=8888891&key=1BAn8AY2sg49i&format=frame)**__** and/or **__**[mutation analysis](http://www.genetests.org/servlet/access?qry=140&db=genestar&fcn=term&gtreport2=true&id=8888891&key=1BAn8AY2sg49i&format=frame)**__**, it is possible to identify both **_***IDUA***** mutations in **_**95%**_** of individuals with MPS I.**

## MPS 1 – Biochemical TestingProband

**Glycosaminoglycan (GAG) urinary excretion. **
**Measurement of urinary GAGs may be quantitative or qualitative. **
**Neither method can diagnose a specific lysosomal enzyme deficiency but indicate the likely presence of MPS disorder.**
**Both methods have reduced sensitivity (dilute urine). Qualitative testing can exclude / include certain disorders, but definitive diagnosis requires enzyme determination. **
**Diagnosis of MPS I relies on the demonstration of deficient activity of the enzyme  - L-iduronidase.**
**L-iduronidase activity - measured in most tissues**
**Usually peripheral blood leukocytes, cultured fibroblasts, or plasma.**

## MPS 1 – Family

**Carrier testing for MPS I is **_**COMPLEX.**_
**Relatives of a proband. **
**Carrier testing of immediate family members of affected individuals requires testing of obligate carriers first to determine if their levels of IDUA enzyme activity are clearly distinguishable from normal. The same analysis can then be applied to other family members at risk. **
**General population **
**The interpretation of  -L-iduronidase enzyme activity within the general population is difficult.**
**Overlap exists between the lower end of the normal range of enzyme activity and the upper end of the heterozygous range. **
**Pseudodeficiency alleles for L-iduronidase enzyme activity difficult. **
**Pseudodeficiency relates to the finding of reduced or undetectable IDUA enzymatic activity - artificial substrates.**
**No evidence of altered glycosaminoglycan metabolism with the use of radiolabelled (35S)-GAG. **
**Molecular basis and frequency of pseudodeficiency are unknown.**

## MPS 1 – Mutation Detection

## Glycogen Storage Disease Type 1Proband

**Diagnosis is based on clinical presentation; abnormal blood/plasma concentrations of glucose, lactate, uric acid, triglycerides and lipid; **_**AND **_**molecular testing.**

## Glycogen Storgae Disease Type 1Proband

**Glucagon or epinephrine administration. **
**The lack of either glucose-6-phosphatase catalytic activity or glucose-6-phosphate translocase activity in the liver results in the following:**
**Hypoglycemia: Fasting blood glucose concentration <60 mg/dL **
**Lactic acidosis: Blood lactate >2.5 mmol/L**
**Hyperuricemia: Blood uric acid >5.0 mg/dL **
**Hyperlipidemia: **
**Triglycerides: >250 mg/dL Hypertriglyceridemia- plasma appears "milky." **
**Cholesterol: >200 mg/dL**
**Hepatic enzyme activity.   Snap-frozen liver biopsy - dry ice - overnight delivery. **
**Glucose-6-phosphatase (G6Pase) catalytic activity. **
**In most individuals with GSD type Ia, the G6Pase enzyme activity is lower than 10% of normal. **
**In rare individuals with higher residual enzyme activity, the G6Pase enzyme activity could be higher **
**Glucose-6-phosphate translocase (transporter) activity. G6P translocase activity in vitro is difficult to measure in frozen liver**
**Fresh (unfrozen) liver is often needed and most laboratories refrain from offering this assay. **

## Glycogen Storage Disease Type 1Proband

**The two genes associated with GSDI are:**
***G6PC***** (GSDIa),  about 80% of GSDI.**
***SLC37A4***** (GSDIb), about 20% of GSDI.**
**Molecular testing of *****G6PC***** and *****SLC37A4***** is available.**

## GSD 1 – Mutation Detection

## Glycogen Storage Disease 1Family

**Carrier testing using molecular genetic diagnosis for at-risk family members is available once the mutations have been IDENTIFIED in the proband. **
**Biochemical testing.  Enzymatic testing is **_**UNRELIABLE**_** for use in carrier detection.**

## Glycine EncephalopathyProband

**Glycine encephalopathy is suspected with elevated glycine concentration in urine. **
**An increased CSF-to-plasma glycine ratio suggests the diagnosis.**
**Reliable enzymatic confirmation requires measurement of glycine cleavage enzyme (GCS) activity of liver obtained by BIOPSY or AUTOPSY. **
**Most affected individuals have no detectable activity; **_**testing is available on a research basis only. **_

## Glycine EncephalopathyProband

**The *****GLDC***** gene accounts for disease in 80% of individuals with NKH.**
**The *****AMT***** gene accounts for disease in 10-15% of individuals with NKH.**
**The *****GCSH***** gene accounts for ?**
**Combined mutation detection rate is 60% to 90%.**

## Glycine EncephalopathyProband

## Glycine EncephalopathyProband 

## PKUProband / Family

## PKU Family

**At-risk family members have the option of being tested for carrier status. **
**Partners of carriers have **_**limited **_**access to testing.**_** **_
**Three methods can be used: **
**Molecular genetic testing for at-risk family members; once the mutations have been identified in the proband. **
**Linkage analysis, for the few families in which the mutation has not been identified, if the family is informative for markers within the *****PAH***** gene. **
**Biochemical analysis for determination of carrier status ONLY IF MOLECULAR IS NOT POSSIBLE.**

## PKU Family

**Biochemical analysis can be used to determine carrier status if molecular testing is not possible.**
**Testing relies on analysis of the plasma phe concentration and phe/tyr ratio. **
**This test must take into account circadian variation (i.e., it must be performed before noon after a normal breakfast) and variations in a woman's menstrual cycle; it is not accurate during pregnancy.  **

## PKU – Family

**Prenatal diagnosis is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis or chorionic villus sampling (CVS). **
**Both disease-causing alleles of an affected family member MUST BE IDENTIFIED or linkage established in the family.**