DMD exon deletion: Deletions in male patients are detected by multiplex PCR, screening the most commonly involved exons.

DMD carrier test: Females with a documented mutation (required) in a male relative are screened in a semi-quantitative PCR assay.

DMD linkage: For families without an identified mutation, linkage analysis can be performed, using intragenic polymorphic markers.

Gaucher Disease

Gaucher Disease is a multisystemic disorder that mainly affects liver, spleen, bone marrow, and/or nervous system. There is a continuum of clinical findings, ranging from a perinatal-lethal form, to early or late CNS involvement with varying progression, to an asymptomatic form. An estimated 1:18 individuals of Ashkenazi Jewish descent are carriers. GD is caused by deficiency of the enzyme beta-glucocerebrosidase. The GBA gene (chromosomal locus 1q21) consists of 11 exons spanning over 7 kb. Our laboratory tests for the 4 most common Ashkenazi Jewish mutations (N370S, IVS2nt1g>a, 84GG, and L444P) with a detection rate of 95%.

HEREDITARY FRUCTOSE INTOLERANCE
(PCR & ASO)

Some segments of the human genome exhibit polymorphism due to a variable number of tandem repeats (VNTR). These polymorphic regions are amplified from DNA extracted from blood samples from both parents and patients, or mother of the fetus.

LCHAD deficiency is an autosomal recessive condition resulting from the deficiency of long-chain 3-hydroxyacyl-Co A dehydrogenase, which is normally involved in mitochondrial fatty acid b-oxidation.  Patients with LCHAD deficiency often present with lethargy, hypoglycemia, hypoketotic hypoglycemia, cardiomyopathy, hepatopathy, retinopathy, hypotonia, peripheral neuropathy or Reye-like syndrome.  The diagnosis of fatty acid b-oxidation defects, such as LCHAD deficiency, is often complicated by the unremarkable findings in biochemical screening tests, especially between metabolic crises.  In such cases, genetic testing could be very informative.

The LCHAD gene, on chromosome 2p23, encodes the a subunit of the mitochondrial trifunctional protein involved in fatty acid b-oxidation. Our laboratory analyzes the most common mutation of the a subunit, G1528C (c.1658G>C in the mRNA, NM_000182). This alteration changes amino acid 474 in the mature peptide (p. Glu474Gln), and is detected by PCR/restriction digestion. It is detected in >83% of patients (IJlst, et al, 1994; Sims et al, 1995; Tyni et al, 1997).

MCAD

MCAD is the most common enzyme deficiency in fatty acid oxidation. The condition usually presents with episodic hypoketotic hypoglycemia provoked by fasting or infection in the first 2 years of life. MCAD may also present clinically as an acute encephalopathy. Between episodes, patients with MCAD deficiency can appear normal, but in some cases, the first episode is fatal and can resemble sudden infant death syndrome (SIDS).

MCAD is an autosomal recessive disorder and the ACADM gene has been localized to chromosome 1p31. The A985G mutation (amino acid substitution K329E in the precursor protein) accounts for more than 90% of deficient alleles, with an estimated carrier frequency of 1:68 in Northwestern Europe.

Mitochondrial DNA Panels I, II-Regular, II-Expanded, LHON:

The unique pathogenetic features of mtDNA include maternal inheritance, heteroplasmy, and threshold effect for clinical expression, high spontaneous mutation rate and mitotic segregation. The mitochondrial diseases are considered complex and difficult to diagnose due to their wide variation in phenotypic expression and considerable clinical overlap between syndromes. The syndromes below are associated in most cases with specific alterations in mitochondrial DNA (mtDNA): large deletions or duplications/rearrangements in Kearns-Sayre Syndrome (KSS), Progressive External Ophthalmoplegia (PEO), and Pearson’s marrow syndrome; multiple deletions in MNGIE (Myo-Neuro-Gastro-Intestinal Encephalomyopathy); point mutations in Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS); Myoclonic Epilepsy and Ragged Red Fibers (MERRF); Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP); Leber's Hereditary Optic Neuropathy (LHON); Leigh syndrome; Familial Bilateral Striatal Necrosis (FBSN); cardiomyopathy, diabetes; deafness; maternally inherited myopathy; multisystem disorders.

Mitochondrial DNA Panel I: Multiplex PCR/ASO is used to detect 9 point mutations: A3243G (MELAS, diabetes/deafness), T3271C (MELAS); A8344G (MERRF); G8363A (cardiomyopathy, MERRF, hearing loss); T8993C, T8993G (NARP, Leigh’s); T9176C (Leigh’s, FBSN); G13513A (Leigh’s, MELAS); T14709C (myopathy, cerebellar ataxia, diabetes/deafness, mental retardation).

Mitochondrial Panel II-Regular: The laboratory’s routine Mitochondrial DNA Panel 2-Regular analysis uses Southern blot hybridization to detect mtDNA deletions. The analysis does not always detect duplications and rearrangements. Please provide additional information if screening for duplications is clinically indicated, and order Panel 2-Expanded.

Mitochondrial Panel II-Expanded: detects deletions and duplications/rearrangements, using additional Southern blot analysis; recommended for KSS, chronic PEO, Pearson’s marrow syndrome.

LHON Panel: Multiplex PCR/ASO is used to detect the 3 most common LHON mutations that occur in ~95% of patients: G3460A, G11778A, and T14484C.

Phenylketonuria (PKU), an autosomal recessive disease, is the most common inborn error of amino acid metabolism. The lack of phenylalanine hydroxylase (PAH) activity causes persistent hyperphenylalaninemia, with irreversible impairment of brain development and severe mental retardation in untreated children. The PAH gene, located on 12q22-24, spans over 90 kb and consists of 13 exons. Approximately 400 mutations have been reported, which are spread throughout the gene and vary among populations.

PKU-Panel: Our laboratory tests for 15 PAH mutations with an average detection rate of ~50%. Included are: F39L, L48S, I65T, R158Q, R243X, V245A, R261Q, E280K, P281L, A403V, R408Q, R408W, Y414C, IVS10 nt-11G>A, and IVS12 nt1G>A. (Classical mutations, with <1% enzyme activity in vitro, are underlined.)

For efficiency purposes, we perform the PKU-Panel as a batch test, for three or more patients.

PKU-Seq: Up to 13 exons and exon-intron junctions of the PAH gene are sequenced to identify two mutations in patients, with a >95% detection rate. Parental blood specimens are requested as mutation controls (but their results are not reported).

We also offer a two-tiered option, with an initial screen for the common mutations in the PKU-Panel. The test order is changed to PKU-Panel if two mutations are identified; if we do not identify two mutations, we proceed with PKU-Sequencing.

PKU-Direct: Familial mutations are screened to determine carrier/affected status of relatives. For prenatal testing, parental samples are required to rule out maternal cell contamination and confirm relatedness.

PKU-Linkage: In the absence of a known mutation, linkage analysis using intragenic polymorphic markers can be used to trace the transmission of the mutant PAH gene in informative PKU families with one or more affected family members. This method can be applied to prenatal and carrier tests.

Methylation analysis using PW71 or SNRPN probes is a reliable diagnostic test (ASHG/ACMG report Am J Hum Genet 58:1085-8, 1996), detecting ~99% of PWS and 75-80% of Angelman syndrome.

Spinal Muscular Atrophy:

Spinal Muscular Atrophy (SMA) is the second most common lethal, autosomal recessive disease in Caucasians. SMA affects about 1:10,000 live births and has a carrier frequency of 1:40 - 1:60. This neuromuscular disease is characterized by the degeneration of the alpha motor neurons of the spinal cord, which leads to progressive symmetrical weakness and wasting in the proximal muscles. Three types of SMA have been distinguished on the basis of clinical severity and age of onset: SMA type I (Werdnig-Hoffmann disease): most severe phenotype - patient cannot sit unaided and usually dies in infancy before age 2; SMA type II (intermediate): onset in early childhood - often results in early impairment of walking; and SMA type III (Kugelberg-Welander disease): onset in late childhood - considerable clinical heterogeneity and the risk of wheel chair dependence in adult years.  

The Survival Motor Neuron (SMN1) gene that is responsible for all three types of SMA has been mapped to chromosome 5q13.  Although all three have in common the loss of SMN1 exon 7, the differences in severity are likely attributable to contiguous gene deletions, type of mutation (deletion, rearrangement, gene conversion), and modifying genes. Notably, there is an inverse correlation between disease severity and the number of copies of the adjacent homologous gene, SMN2. 

The deletion, absence or interruption of both copies of SMN1 can be used to confirm the clinical diagnosis using PCR/ASO hybridization. Because the majority of SMA carriers have only one copy of SMN1 exon 7, they can be identified by gene dosage analysis in a semi-quantitative PCR test.

Direct mutation analysis: Presence or absence of exon 7 and 8 of the SMN1 gene is analyzed using PCR-ASO hybridization.

SMA carrier test: Dosage of SMN1 exon 7 is analyzed by semi-quantitative PCR assay.

SMA linkage: For families with only one identified mutation or for prenatal SMA tests with negative results, linkage analysis is recommended. The haplotypes for microsatellite repeats in the 5q13 region are compared in the fetus and proband.

Ashkenazi Jewish Panel: Canavan Disease, Cystic Fibrosis, Familial Dysautonomia, Tay-Sachs Disease.  Point mutation detection. 

Canavan Disease (CD) is a neurodegenerative disease resulting from deficiency of the enzyme aspartoacylase. The condition is characterized by severe mental retardation, large head size, blindness, hypotonia, seizures, and spongy degeneration of the brain white matter. The average life span of affected infants is 18 months. The carrier rate among Ashkenazi Jews is ~1:40. The ASPA gene encoding aspartoacylase (chromosomal locus 17pter-p13) consists of 6 exons spanning over 29 kb. Our laboratory tests for the two most common mutations, Y231X and E285A, which account for 98% of Canavan disease mutations among Ashkenazi Jewish patients. 

Cystic Fibrosis (CF) is a chronic multisystemic disease that affects the lungs, digestive tract, reproductive system, and sweat glands. Symptoms may include failure to thrive, chronic lung infection, recurrent pneumonia, and excessive mucus production. An estimated 1:29 individuals of Ashkenazi Jewish descent are carriers. The CFTR gene (cystic fibrosis transmembrane conductance regulator) maps to chromosomal region 7q31.2, and consists of 27 exons spanning over 230 kb. More than 900 pathological mutations have been reported. Our laboratory tests for the 23 mutations recommended by the American College of Medical Genetics, which include the 5 most common Ashkenazi Jewish mutations (delF508, G542X, W1282X, N1303K, and 3849+10kb C>T), for which this test has a 95% detection rate.  

Familial Dysautonomia (FD) is an autosomal recessive disorder with progressive degeneration of the sensory and autonomic nervous systems. Symptoms include lack of tearing, excessive sweating, emotional instability, episodic vomiting, insensitivity to pain and temperature, postural hypertension, and poor motor coordination. Pulmonary and cardiac complications are the common causes of death, with ~50% survival to age 30. The estimated carrier frequency is 1:30 Ashkenazi Jews. The IKBKAP gene (chromosomal locus 9q31) encodes IkB kinase complex-associated protein. Our laboratory tests for two mutations: IVS20 nt6T>C (homozygous in 99% of patients of Ashkenazi Jewish descent) and R696P. 

Tay-Sachs Disease (TSD) is a progressive neurodegenerative disorder resulting from deficiency of the enzyme, Hexosaminidase A. Acute infantile TSD is characterized by an exaggerated startle reaction, cherry-red macula, hypotonia, seizures, blindness and dementia, with rapid progression and death by age 4. Juvenile TSD patients survive to late childhood/adolescence, while chronic/adult onset TSD patients have muscle weakness and CNS symptoms and/or psychosis. The carrier rate among Ashkenazi Jews is ~1:25. The HEXA gene (chromosomal locus 15q23-q24) consists of 14 exons spanning 35 kb. Our laboratory tests for the three most common Ashkenazi Jewish mutations (1278+TATC, IVS12nt1g>c, G269S) with a detection rate of 97%.

Factor V Leiden disease:  The factor V Leiden R506Q mutation, which causes resistance to activated protein C (APC), is the most frequent risk factor of inherited thrombosis in Caucasians, accounting for over 90% of the APC resistance cases and is inherited as an autosomal dominant trait.

Methylenetetrahydrofolate Reductase (MTHFR):  Homozygosity of the MTHFR 677C>T variant is associated with elevated plasma homocysteine levels and increased risk of thrombosis. Recent studies indicated that neural tube defects and unexplained recurrent embryo losses in early pregnancy are also associated with hyperhomocysteinemia and MTHFR variants.

Prothrombin:  The prothrombin 20210G>A mutation, associated with elevated plasma prothrombin levels, also increases the risk of venous thrombosis. The risk of being affected with deep vein thrombosis, pulmonary embolism, and cerebral-vein thrombosis is approximately 3-10 higher in carriers with factor V Leiden and/or prothrombin gene mutation than non-carriers. The carrier frequencies for 20210G>A in Caucasians is 1-2% but lower in other ethnic groups.