TSD is a neurodegenerative disorder resulting from deficiency of the lysosomal enzyme hexosaminidase A (HEX A), resulting in accumulation of the cell membrane glycolipid G_M2 ganglioside within lysosomes (for review, see Refs. 14, 15). The clinical course of TSD is characterized by normal development for the first few months of life followed by progressive loss of motor skills, macrocephaly, seizures, blindness, and death usually before 4 years of age. Infantile TSD always is fatal and there is no effective treatment. Later-onset forms of TSD have slower disease progression. TSD is an autosomal recessive disease and has a carrier frequency of approximately 1 in 30 among Ashkenazi Jewish individuals and 1 in 250–300 in most other populations. Genetically isolated populations such as the French Canadians of Quebec, Cajuns from Louisiana, and the Amish in Pennsylvania also have carrier frequencies similar to the Ashkenazi Jews. The first carrier screening programs began in 1970 and used the measurement of HEX A activity in serum, leukocytes, or tears. When the HEXA gene encoding HEX A was cloned in 1987, disease-associated mutations were identified. Current testing for TSD utilizes both biochemical and molecular testing by various methods.

Carrier screening for TSD, which began in the 1970s and was later endorsed by the American College of Obstetricians and Gynecologists (ACOG) and the ACMG, has been a model for population screening programs. As a result, there has been a 90 % reduction in the incidence of TSD in the North American Ashkenazi Jewish population, such that the incidence of TSD is now three- to fourfold higher in non-Jews by comparison.

A pseudodeficiency allele, p.R247W, is present in approximately 2 % of Ashkenazi Jewish individuals who are carriers by the enzymatic assay. The p.R247W variant decreases the activity of HEX A for the artificial substrate used in the laboratory but does not cause TSD since it does not affect HEX A activity for its natural substrate, GM2 ganglioside. About 36 % of non-Jewish individuals who are carriers by enzyme analysis have a pseudodeficiency allele (32 % p.R247W and 4 % p.R249W). In addition, screening for the three common Ashkenazi Jewish mutations and an additional mutation (c.1073+1G>A; IVS9) will identify approximately 95 % of Ashkenazi Jewish carriers, but only 40–50 % of disease-causing alleles in the non-Ashkenazi Jewish populations. Other populations who are at high risk, such as the French Canadians, Cajuns, and Pennsylvania Dutch, have their own common alleles. Therefore, the mutations included for population screening must target the specific mutations of the ethnic background of the individuals being tested.

GD is another prevalent autosomal recessive lysosomal storage disorder that is found with higher incidence in the Ashkenazi Jewish population. The carrier frequency is 1 in 15 in the Ashkenazi Jewish population and 1 in 100 in other populations [16] (for comprehensive review on GD, see Ref. 17). A defect in the enzyme glucocerebrosidase leads to the accumulation of glucocerebrosides in lysosomal compartments in macrophage/monocyte-derived cells, particularly in the liver, bone marrow, spleen, and lung. Several forms of GD exist. Type 1 GD has a wide range of clinical presentations, from asymptomatic to symptoms including bone disease, hepatosplenomegaly, anemia, and thrombocytopenia, but without primary central nervous system involvement. Types 2 and 3 have primary central nervous system involvement that varies by age of onset and rate of disease progression. Type 2 GD patients usually have an earlier age of onset than type 3 patients, with acute disease progression and death by approximately 2 years of age. Type 3 patients have onset in early childhood to adolescence and survive into their first to fourth decade of life. A perinatal-lethal form of GD also can occur, as well as a cardiovascular form characterized by aortic and mitral valve calcification, ophthalmologic abnormalities, and hydrocephalus.

The glucocerebrosidase gene (GBA) and its transcribed pseudogene (ψGBA) are located on chromosome 1q21. Approximately 200 mutations causing GD have been identified. Many of the mutations are most likely due to gene conversion events with the pseudogene.

The demonstration of deficient glucocerebrosidase activity in leukocytes establishes a diagnosis of GD but is unreliable for carrier detection. Therefore, molecular testing is useful for carrier identification, prenatal testing, and genetic counseling.

Four mutations (p.N409S, c.115+1G>A, c.84dupG, p.L483P) are responsible for approximately 95 % of disease-causing alleles in Ashkenazi Jewish individuals and 50 % of disease-causing alleles in non-Ashkenazi Jewish individuals [16]. Most clinical molecular laboratories performing GD molecular testing assess at least these four mutations. Several factors must be considered when designing molecular testing for GD. Primers must be selected that avoid amplification of the pseudogene which is located 16 kb downstream and is approximately 96 % identical to the functional gene. Recombinant alleles, which are thought to result from unequal crossovers between exons 9 and 10 of the functional gene and pseudogene, contain two or more point mutations, including p.L483P. If p.L483P alone is tested, misdesignation of the genotype may occur. This may be important, as the recombinant allele is typically associated with a more severe genotype. Mistyping also is possible when a 55 base pair (bp) deletion in exon 9 (c.1263_1317del), is present in combination with the common p.N409S allele. Homozygosity of p.N409S would be observed even though the true genotype is p.N409S/55 bp deletion. Therefore, the 55 bp deletion should be analyzed in patients who are found to be homozygous for p.N409S.

Genotype-phenotype correlations have been widely investigated in GD [18]. While overlaps occur, some generalizations can be made. The presence of a p.N409S allele is predictive of type 1 disease. Individuals with p.L483P in the presence of a null allele will usually have type 2 GD, while homozygosity for p.L483P typically results in type 3 GD. Homozygosity for the p.D448H allele has been associated with the rarer cardiovascular form of GD.

Galactosemia is an autosomal recessive disorder caused by deficient or absent activity of one of three enzymes involved in the metabolic pathway to convert galactose to glucose: galactokinase (GALK), galactose-1-phosphate uridyl transferase (GALT), and UDP-galactose 4′-epimerase (GALE). Classic galactosemia is due to a severe reduction or absence of the GALT enzyme, and has an incidence of 1 in 40,000–60,000 in European newborns (for review, see Ref. 19).

The GALT gene is located at 9q13, is about 4 kb in length, and consists of 11 exons. More than 150 mutations in the GALT gene have been reported, most of which are private mutations [20]. The p.Q188R mutation is the most frequent mutation associated with classic galactosemia in many populations, and accounts for 64 % of disease alleles in Europeans, 60–70 % in Americans, and 50–58 % in Mexican Hispanics [20, 21]. Ethnic-specific mutations include p.K285N, p.S135L, c.253-2A>G, and a 5 kb deletion in Caucasian, African-American, Hispanic, and Jewish patients, respectively.

Molecular testing is used for confirmation of diagnosis, carrier detection, prenatal diagnosis, prognosis, and genetic counseling. Because the detection rate of molecular testing is less than 100 % and biochemical testing is highly accurate, mutation detection is carried out in parallel with biochemical analysis.

Prenatal diagnosis for galactosemia can provide the opportunity for immediate dietary restriction of the newborn. Although galactosemia is considered “treatable,” symptoms such as ID, growth delays, speech dyspraxia, abnormal motor function, and premature ovarian failure in women may still occur even with early intervention and lifetime dietary restrictions. These long-term phenotypes often are associated with specific mutations; for example, p.Q188R can cause premature ovarian failure and speech dyspraxia. Genetic counseling is very important for parents of affected fetuses before a choice regarding pregnancy outcome is made.

Initial molecular testing for the diagnosis of galactosemia focuses on the most prevalent mutations or the p.N314D mutation associated with Duarte galactosemia (Duarte-2) by quick and cost-effective methods, such as multiplex PCR followed by restriction enzyme digestion [22]. If only one mutation or no mutations are found, screening of all 11 exons and exon-intron boundaries by DNA sequencing can be performed [22]. A detection rate of 96 % can be achieved by a combination of testing for prevalent mutations and DNA sequencing [23]. Gross deletions and duplications are detected by aCGH or MLPA.

Genotype-phenotype associations have been established for some mutations [20]. For example, p.Q188R, p.K285N, and p.L196P alleles have undetectable GALT activity and are associated with severe phenotypes, whereas p.S135L and p.T138M are less severe and are usually associated with a good prognosis. In addition to the classic form of galactosemia, which has less than 5 % of the normal GALT activity, the Duarte-2 variant associated with the p.N314D variant has 50 % of the normal GALT activity. The p.N314D allele is in linkage disequilibrium with four polymorphisms, c.329-27G>C, c.378-24G>A, c.507+62G>A, and c.-119_-116delGTCA. The symptoms of Duarte-2 patients are mild compared to the classic type, but Duarte-2 still results in long-term phenotypes such as ID and growth delays, speech dyspraxia, abnormal motor function, and premature ovarian failure in women.

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder caused by deficient or absent activity of aldolase B. The aldolases are a group of tetrameric enzymes that are highly conserved and involved in the cleavage of fructose-1,6-biphosphate. Aldolase B also cleaves fructose-1-phosphate, and thus is a key player in fructose metabolism, as well as in gluconeogenesis and glycogenolysis. Aldolase A is found predominantly in muscles, whereas aldolase B is found predominantly in the liver, kidney, and intestine, and aldolase C is predominantly in the brain. Severe reduction or absence of only the aldolase B enzyme causes HFI, and has an incidence of 1 in 20,000–30,000 in European newborns (for review, see Ref. 24).

Typically in HFI, symptoms do not arise until an infant is exposed to fructose, sucrose, or sorbitol-containing foods, usually at the weaning of breast milk and introduction of table foods. Acute ingestion of the offending sugar can result in a variable presentation of abdominal pain, vomiting, progressive liver dysfunction, hypoglycemia, uric and lactic acidosis, and renal tubulopathy, depending on the dose of the sugar ingested, and timing and length of exposure. Chronic, low-dose ingestion of fructose-containing foods can cause a subacute picture of hepatomegaly and failure to thrive. Affected individuals typically have an aversion to sweets. The acute and chronic symptoms are, for the most part, reversible, if a fructose-free diet is initiated. Once on treatment, the prognosis is good with a normal life expectancy.

The ALDOB gene is located at 9q22.3, is about 14 kb in length, and consists of nine exons, eight of which are translated. More than 40 mutations have been identified (http://​www.​bu.​edu/​aldolase/​HFI/​hfidb/​hfidb.​html). The most common allele, p.A149P, accounts for 50–65 % of all mutations in the European population, and together with two other common mutations, p.A174D and p.N334K, account for more than 80 % of mutant alleles in the European population [25]. Most of the missense mutations are located in exons 5 and 9. Studies show that the p.A149P decreases the thermal stability of the enzyme. Figure 11.1 illustrates the locations of the most common variants in the aldolase B protein structure.

Molecular testing is used for confirmation of diagnosis, carrier detection, prenatal diagnosis, and genetic counseling. Enzymatic testing of aldolase B activity can only be performed from liver, kidney, or intestinal tissue. Thus, molecular testing is now the preferred method. Prenatal diagnosis for HFI can provide the opportunity for early dietary restriction and prevention of acute symptoms and liver disease.

Targeted mutation analysis of the common alleles using multiplex PCR is a cost effective and rapid method for the European population [27]. If only one mutation or no mutations are found, DNA sequencing of the entire coding region and exon-intron boundaries should be performed. For those not of European descent, DNA sequencing should be performed as a first-line method.

Genotype-phenotype correlations are not clear. HPI patients with the same genotype have varied presentations and reactions to ingestion of fructose.

Glycogen storage diseases (GSD) are a group of heterogeneous genetic disorders characterized by the accumulation of glycogen in tissues and have an overall incidence of 1 in 20,000–25,000 live births [28]. Fourteen types of GSD that vary significantly in clinical phenotypes, age of onset, and affected organs have been identified (www.​omim.​org). They are caused by defects in one of 18 genes in glycogen metabolism. A summary of the 14 GSD types is presented in Table 11.2. The most common and severe types, GSD I, II, III, and IV, are discussed below.