Hyperglycerolemia (MIM 307030) is also known as glycerol kinase deficiency (GKD) and GK1 deficiency.394 GKD can be subdivided into three clinical forms by phenotype. Complex glycerol kinase deficiency (CGKD), or the infantile form,13,59,332,394,395 is a contiguous gene8-55 syndrome (see Chap. 65) involving not only the GK locus but also the AHC (see Chap. 167) and/or the DMD (see Chap. 216) locus in the Xp21 region. Two phenotypes have isolated involvement of the GK locus—the juvenile form,59,395-399 associated with metabolic and central nervous system instability and deterioration, and the benign, or adult, form,6,59,332,395,400-403 detected incidentally with pseudohypertriglyceridemia.58
It is now clear that DMD, GKD, and AHC are caused by mutations in distinct loci, since the cDNAs for the genes responsible for each of these disorders have been cloned.176-178,394,404,405 The existence of individual genes for the CGKD phenotypes had been previously hypothesized5,7 and was confirmed by observation of patients with phenotypes including isolated forms of GKD without DMD or AHC, isolated AHC without GKD or DMD, and forms with breakpoints between AHC and GK or between GK and DMD.
Contiguous Gene Syndrome Involving AHC, GKD, and DMD.
Two brothers with this phenotype were originally described with GKD in 1977,4 although the association with AHC was not recognized until later.5 To date, the largest group of individuals with CGKD have been described with AHC, GKD, and DMD, and others who died prior to the recognition of this syndrome are suspected to have had this diagnosis.4,5,7,8,11-18,20,21,23-25,27-31,35,37,39,41,42,44,47-55 All but one of the patients were male.7 One additional male, who died in the neonatal period, also had ornithine transcarbamylase deficiency (OTCD) associated with a deletion extending centromerically beyond the DMD locus.9,10
A summary was compiled of the clinical findings in 17 patients with CGKD, of whom 15 had AHC, GKD, and DMD.27 Among these 15 patients, the following features were observed, with frequencies determined from the number of clinical descriptions in which a comment was made about the particular feature: psychomotor retardation (12/12); short stature (10/12); abnormal genitalia (6/13); osteoporosis (6/13); and characteristic facies with strabismus (6/13), wide-set eyes and drooping mouth (4/13), or dysmorphic facies (1/13, the individual who had AHC, GKD, DMD, and OTCD). The abnormal genitalia included anorchia and cryptorchidism, features of gonadotropin deficiency associated with X-linked cytomegalic AHC.8,27,406
The dysmorphic features observed in patients with CGKD, while mild, are characteristic.53 Facial features include the conformation of the lower forehead, eyebrows, and root and bridge of the nose, with hypertelorism and rounded palpebral fissures, described as an “hourglass” midface appearance. Additional facial features typically seen in these boys are esotropia, a down-turned mouth, and flattened ear lobes. Agenesis of the corpus callosum has been reported in a patient with AHC, GKD, and DMD.55
Additional patients have been described with a phenotype involving the AHC and DMD loci without measurement of GK activity or documentation of a GK mutation. A Japanese boy presented with vomiting, weight loss, hyponatremia (116 mEq/liter), and hyperkalemia (7.5 mEq/liter) at 23 days of age.407 Adrenal insufficiency was documented and treated, and an adrenal scintigram showed bilateral absence of the adrenal glands. Serum creatine kinase (CK) was elevated, a muscle biopsy was consistent with DMD, and his clinical course was characteristic of this disorder. Generalized seizures, unconsciousness, and apnea developed after 1 week of insufficient medication, and the patient died at 3 years 5 months of age. Autopsy findings confirmed the adrenal hypoplasia. Two brothers were reported who had adrenal insufficiency and hypoplasia, dystrophic myopathy and elevated serum CK, severe psychomotor retardation, failure to thrive, and megalocornea.408 Family history included an institutionalized older sister with seizures and mental retardation, a brother with encopresis, and a normal sister. GK activity was not measured in these three patients with AHC and dystrophic myopathy, but they would be expected to have GKD, since the gene loci are ordered AHC, GK, DMD.
There is an extremely high frequency of neonatal and early childhood deaths from unrecognized adrenal insufficiency in these families. Since all the documented patients with AHC, GKD, and DMD living beyond the neonatal period have been developmentally delayed, this would suggest that any patient with DMD and developmental delay should have an evaluation of adrenal function. If this phenotype is ascertained in one family member, then collateral relatives should also be pursued and evaluated. Biochemical or cytogenetic evaluation of 30 patients with DMD identified two patients with deletions extending telomeric of the dystrophin locus.15
Contiguous Gene Syndrome Involving AHC and GKD.
The second largest group of patients with CGKD have AHC and GKD without DMD.7,11,14,18,19,21,22,26,29,32-34,36,46,48-50,409 Characteristic features include adrenocortical insufficiency, hyperglycerolemia, and glyceroluria without myopathy. Some patients have had normal genitalia and others have had hypogonadism. Where there is a comment regarding developmental progress, delay is frequently noted, although psychomotor development was reported to be normal in three.21,34,36 Dysmorphic features were described in one boy, including slightly abnormal pinnae, short palpebral fissures, underdeveloped lower jaw, and decreased hip abduction.32 Two patients have presented with wheezing, initially attributed to asthma but subsequently considered a mild form of Addisonian crisis.46 The family histories of these patients and the high frequency of unexplained early deaths indicate that any patient with evidence of GKD, whether or not developmentally delayed, should have an evaluation of adrenal function.
Contiguous Gene Syndrome Involving GKD and DMD.
Two brothers have been described with CGKD involving GKD and DMD without AHC, indicating a breakpoint between the AHC and GK loci.30 Both boys had initial recognition of muscle weakness at 1 year of age. Progressive muscle weakness, elevated serum CK and aldolase activity, and characteristic muscle histology led to the diagnosis of DMD in the older brother. The younger boy also had elevated serum CK values. Both experienced multiple recurring episodes of vomiting beginning at 11 to 12 years of age. The younger boy required hospitalization for intractable vomiting on several occasions, and dehydration, severe metabolic acidemia (arterial pH 7.01, total CO2 2–3 millimolars), and ketonuria were documented. Both boys showed hyperglycerolemia, glyceroluria, and GKD. Both were developmentally delayed, and normal adrenal function and reserve were documented in them by fasting cortisol and ACTH levels and cosyntropin challenges (Sloan HR: personal communication). They were placed on a low-fat, low-glycerol diet and had no subsequent episodes of vomiting or acidemia during the ensuing 9 months (Sloan HR: personal communication). These episodes and the response to dietary restriction of glycerol are similar to the observations in patients with the juvenile form of isolated GKD. The episodic vomiting and acidemia of these patients with GKD and DMD is particularly interesting, since they have normal adrenal function. Patients with CGKD that includes AHC may have similar episodes, and although these may appear to occur at times of adequate adrenocortical replacement therapy, it may be difficult to determine whether these metabolic abnormalities are truly independent of the AHC. However, the similarity of these episodes to those experienced by children with the isolated symptomatic, or juvenile, form of GKD suggests that the episodes are attributable to deletion of the GK locus in patients with complex GKD.
Isolated Glycerol Kinase Deficiency.
There are two clinical subtypes of isolated GKD, referred to previously as the juvenile and the benign, or adult, forms of GKD.59,332,395
Symptomatic Juvenile Form.
The original two unrelated boys with hyperglycerolemia, glyceroluria, and GKD reported with the juvenile form each presented with an initial episode of vomiting, acidemia, and somnolence or stupor, which on occasion progressed to unconsciousness.59,396 The patient reported by Eriksson et al.396 was hospitalized at ages 4, 6, 7, 8.5, and 9 years with fever, vomiting, and diarrhea interpreted as viral gastroenteritis. These episodes were associated with metabolic acidemia: pH 7.2 to 7.31, standard bicarbonate 13.9 to 17.1 mEq/liter, and base deficit 10.5 to 12.9 mEq/liter. On a separate occasion, at 8 years of age, he experienced two grand mal seizures not associated with one of these episodes, for which he was placed on phenytoin. EEG at that time showed rolandic spikes in the central and parietal right hemisphere. Normal studies included serum CK and IV corticotropin stimulation test. Growth and mental development were considered normal. A maternal granduncle had epilepsy. Organic acid analysis revealed glyceroluria.
The second patient59 presented at 4 years 2 months of age with vomiting, acidemia, hypotonia, fever, and unresponsiveness after ingestion of mouthwash. This episode and those at 4 years 10 months and at 5 years were associated with pH 7.01 to 7.32 and bicarbonate 3.0 to 3.5 mEq/liter. Ketonuria was documented during one episode. Physical and neurologic exams were normal at 6 years 4 months. EEG was normal at 6 years 4 months and at 8 years, except for a single 1-s burst of diffuse bilateral polyspike and wave discharge. A muscle biopsy examined by light and electron microscopy was remarkable only for increased numbers of morphologically normal mitochondria. Serum CK, cortisol, and ACTH were normal. Intelligence was above average at age 7 years, with IQ of 145 and 122 by two different tests. Pseudohypertriglyceridemia led to his diagnosis. A low-fat diet (<30 percent of total calories) was associated with an absence of further episodes at the time of the report, but subsequent episodes apparently were associated with dietary indiscretions (unpublished), suggesting that the episodes might be related to glycerol ingestion. Family history included hypertriglyceridemia.
A third patient was diagnosed with isolated GKD at 2 years of age but in retrospect had presented initially with episodic hypothermia and lethargy beginning in the first week of life.397 Temperatures were as low as 94°F, and at 18 months of age an episode was preceded by vomiting and diarrhea beginning 2 days before lethargy and hypothermia. He was developmentally delayed, with a development quotient (DQ) of 69 at 26.5 months of age. He was placed on a low-fat diet and experienced no subsequent episodes. His development was reported to be improving, and at 3.5 years his IQ was 94. His overall performance was considered to be within normal limits, but he did exhibit mild attention problems and slightly delayed fine motor coordination, attributed to mild ataxia.
Bonham and Crawford briefly described a fourth patient who presented at 6 years of age with a Reye syndrome-like illness and hypoglycemia to 0.6 millimolar.398 They argued that the hypoglycemia resulted from disruption of glucose homeostasis by interruption of glycerol metabolism. In addition, they proposed that the distinction between the symptomatic juvenile and benign adult forms of GKD may be only that those patients with the benign clinical course have not been challenged metabolically398 (and Bonham JR: personal communication). According to this hypothesis one might expect pedigrees containing individuals with acute episodes as well as those with benign courses. Observations have been extremely limited, and family members with benign courses could be underascertained. Alternatively, the phenotypic distinction between the symptomatic and benign forms of GKD may not be so discrete, as will be discussed below in the section “Genetics.”
A fifth patient presented as a neonate with hypotonia, and at age 5 weeks he had apnea and cyanosis requiring stimulation.399 After another apnea episode at age 5 months and an examination showing persistent truncal hypotonia and symmetrically increased lower extremity deep tendon reflexes, brain imaging revealed mild communicating hydrocephalus, EEG was normal, and the boy exhibited glyceroluria and hyperglycerolemia. His dietary fat was reduced to approximately 20 percent of energy intake until he was 15 months old. By 2 years of age clinical signs had resolved, and repeat MRI at age 2 years 5 months revealed normalization of the earlier abnormalities, with only mild prominence of the lateral ventricles. The authors suggested that the dietary change was unrelated to the resolution of the structural CNS abnormalities and urged consideration of GKD in patients with apnea, hypotonia, mild developmental delay, and/or brain imaging abnormalities.
The resemblance of the acute episodes experienced by patients with the symptomatic, isolated form of GKD (particularly the original two patients59,396) and by those with glycerol intolerance (see “Glycerol Intolerance Syndrome” below) is intriguing. These episodes appear to be reduced by restricted fat, and hence glycerol, intake. Patients are susceptible to these acute spells at times of intercurrent illnesses, presumably as a consequence of catabolism and breakdown of fat, with liberation of glycerol. IV glucose during fasting and catabolism imposed by intercurrent illness may prevent these spells. These observations indicate that acute episodes of vomiting, acidemia, and progressive lethargy, at times associated with hypothermia and ketosis, are a feature of symptomatic GKD, whether isolated or part of a contiguous gene syndrome.
Families with the benign, or adult, GKD phenotype are typically ascertained incidentally when a male proband undergoes blood lipid screening that reveals pseudohypertriglyceridemia.6,58,400-403 Hyperglycerolemia was noted in a mother of three GK-deficient sons in one of these families,401 and the daughter of one of the men had intermediate GK activity.58 Hyperglycerolemia is subsequently recognized, because the apparent hypertriglyceridemia is not consistent with the rest of the workup for hyperlipidemia or because the individual does not respond as expected to hyperlipidemia management. The men with this biochemical abnormality ranged in age up to 76 years.
Associated medical problems among those with benign GKD included mild diabetes mellitus,6,401 myocardial infarctions,6 laryngeal carcinoma in situ, 6 osteoarthritis,6 herpes zoster ophthalmicus,6 diarrhea,401 and a positive family history of diabetes mellitus.6 Two probands were discovered during routine medical evaluation and were in good health.58,400 This was in an older population, and while workup of these patients for myopathy and adrenal function was not described, there was no clinical evidence of these features.
Patients with adrenal insufficiency consequent to contiguous gene deletion must be treated in the same way as those with isolated AHC (see Chap. 167). This requires replacement doses of a glucocorticoid, such as hydrocortisone, and therapeutic doses of a mineralocorticoid, such as fludrocortisone (Florinef). Patients who have died with these AHC-associated phenotypes died prior to diagnosis or at times when steroid doses had been reduced to subtherapeutic ranges. hypogonadotropic hypogonadism (HH) has been treated with testosterone to promote development of secondary sexual characteristics.33
Patients with dystrophic myopathy require supportive treatment. It must be noted that not all these patients have the classic DMD phenotype; early counseling and treatment must recognize that the course of the myopathy is variable and may be mild.
Individuals with GKD, either isolated or part of a contiguous gene syndrome, who have been placed on a low-fat (i.e., low-glycerol) diet have had elimination or reduction of subsequent episodes. Therefore, dietary restriction of glycerol should be considered in patients with GKD and episodic vomiting and acidemia, but it must be recognized that these symptoms may also be attributable to adrenal insufficiency. IV glucose during fasting and catabolism associated with intercurrent illnesses can prevent episodes at these times.
The key to the treatment of these patients is careful documentation of the phenotype. Individuals with the benign form of isolated GKD have required no intervention, although the question has been raised whether metabolic stress might precipitate symptoms in them as well.398 To date, however, no observations of symptomatic episodes have been reported in individuals or pedigrees with benign isolated GKD.
All patients with GKD have shown evidence of hyperglycerolemia and glyceroluria. The glyceroluria frequently comes to attention during a general metabolic evaluation, which includes urine organic acid analysis by GC/MS. The glyceroluria is substantial in these patients, leading to contamination of the organic acid fraction by the neutral compound, glycerol, when a solvent extraction procedure is used.4,56 If ion exchange chromatography is used in preparation of the specimen for GC/MS,56,57 glycerol may not be seen with the organic acids but will be present in the neutral fraction.4 Even using solvent extraction procedures, at least two individuals with GKD have been reported with negative initial urine screens for glycerol.25,396 If glyceroluria is suspected, it is preferable for the GC/MS screen to be performed in a laboratory that has had previous experience in the evaluation of such patients. Quantitation of the urinary concentrations of glycerol in these patients has resulted in values of 41 to 345 millimolars (normal, ≤0.2 millimolars),7,8,14-16,26-28,34,396,399,410 90 to 193 mmol/mmol creatinine55 (normal, not detectable),25,411 or 11 to 360 mmol/24 h (normal, <1 mmol/24 h).6,32,58,400,401
Hyperglycerolemia may come to attention during evaluation of pseudohypertriglyceridemia,58 which in fact represents elevated free glycerol in the blood (see “Introduction and Historical Perspective”). Different methods of serum triglyceride measurement may give discrepant results, depending on the method used. The routine clinical laboratory method measures glycerol released after lipolysis; an alternative method relying on solvent extraction and colorimetry does not show interference by water-soluble compounds such as free glycerol.6 The hyperglycerolemia in individuals with pseudohypertriglyceridemia has been measured in plasma and serum and has ranged from 1.8 to 8.3 millimolars (normal, 0.02 to 0.27 millimolars).4,7,8,15,16,23,26-28,30,34,55,58,59,396,399-401,410 No differences in degree of glyceroluria or hyperglycerolemia were noted between the different phenotypes described above (“Clinical Aspects”), and the values may vary considerably even within the same individual.
Deficiency of GK activity has been documented in a number of tissues, including intact and disrupted leukocytes,4,6,8,22,23,27,28,31,33,58,107,396,401 liver,5,31,107,400 kidney,5,107 small intestine,5 adrenal gland,31 intact and disrupted fibroblasts,5,7,8,14,16,21,22,25,27,30,33,59,98,107,396 Epstein-Barr-virus-transformed lymphoblastoid cell lines,21 and cultured amniotic fluid cells.19,32 These have been assayed using radiochemical or spectrophotometric methods to measure glycerol conversion to glycerol 3-phosphate, to CO2, to protein, or to phosphoglycerides and triacylglycerols. The radiochemical assay of glycerol GK activity4,5,21,107 has been superior to the spectrophotometric assay in our hands. Because of the variability of this assay, some may find it helpful to measure the incorporation of labeled glycerol into trichloroacetic acid precipitable counts in situ in intact cells21,25,27,59,107 in parallel with the in vitro assay. Incorporation of labeled glycerol into glycerolipids also distinguishes patients from controls.98 Biochemical methods should be supplemented with molecular genetic diagnostic studies (see “Genetics” below).
Prenatal diagnosis has been successfully performed for GKD. One patient with a deletion resulting in AHC and GKD was diagnosed in utero using maternal estriol measurements before GKD was recognized in his family,412 and two pregnancies at risk for CGKD with AHC, GKD, and DMD were diagnosed as normal using this approach.413 GK activity is present in amniocytes.413 A 26-year-old woman with a son who had died at 12 months of age with the CGKD phenotype, including AHC, GKD, and DMD, underwent amniocentesis at 18 weeks of pregnancy.19,32 Radiochemical assay of GK activity in homogenates of cultured amniocytes revealed the enzyme deficiency. Total amniotic fluid glycerol (free and triglyceride bound) was 1.740±0.037 millimolar, which was 9.0 SD above the control mean and twice the highest control concentration. Analysis of DNA from cultured amniotic fluid cells and the aborted fetus confirmed the diagnosis of an affected fetus. Prenatal diagnosis performed in another pregnancy at risk for CGKD with AHC and GKD relied on amniotic fluid glycerol and maternal plasma estriol concentrations.34 The limited genetic probes done in this region at that time did not detect a deletion or an informative polymorphism.34 Exclusion of the CGKD in a fetus at risk for AHC, GKD, DMD, and OTC was performed using molecular genetic techniques.10 These studies show that there are several different and complementary approaches to prenatal diagnosis of GKD.
The GK gene maps to the Xp21 region, where the locus order is Xpter– AHC - GKD - DMD –cen (Fig. 97-5). See GenBank X78211 for genomic DNA and pseudogenes. This gene order was initially based on the clinical observation that if only two of these loci are involved, the patients have either AHC and GKD or GKD and DMD, but no patient has been described with AHC and DMD without GKD. Where sublocalization has been possible cytogenetically, it would appear that these deletions involve Xp21.3-p21.2.
Summary of clinical phenotypes involving the loci surrounding GK . The patients' phenotypes allowed ordering of the loci as shown. MR represents a separate locus causing mental retardation, while (MR)DMD represents the mental retardation sometimes seen in DMD patients. Some patients have MR, AHC, GK, and DMD. Some patients have MR, AHC, and GK. Others have AHC and GK, or GK and DMD. Still others have isolated MR, isolated AHC, isolated GK, or isolated DMD. Some DMD patients have associated MR.
An approximate scale for the genomic map in this region was developed by estimation of patient deletion sizes using bivariate flow karyotyping and correlating these results with clinical and molecular genetic data (Fig. 97-6).49 Data derived from PFGE,40,414 YACs,50,415,416 and subsequent identification of additional genes and markers in this region are consistent with this map (Fig. 97-7). These results indicate that AHC and GK are quite close physically, since the YB deletion affecting both loci was undetectable by these methods, implying it was 1 Mb or less in size. The results also show that AHC and GK are within 1 to 2 Mb of the telomeric end of DMD. These distance estimates are consistent with the observations that patients with CGKD are most likely to have the phenotype of AHC, GKD, and DMD, with deletion of all three loci, and that they are least likely to have the GKD and DMD phenotype, with the telomeric breakpoint between the AHC and GK loci.
Estimates of deletion sizes among patients with Xp21 contiguous gene syndromes correlated with clinical and molecular genetic data. The sizes of the patients' deletions were estimated using bivariate flow karyotyping and are represented by the vertical bars with the boundary between the open and lighter hatched sections indicating the mean estimate. The sizes of the mean estimates in megabases are shown above the bars and below the letter designations for the patients. The variance was considered to be ±1 Mb. Breakpoints “anchored” within the DMD locus are shown by double-headed arrows. Loci appear on the left, with numbers to the left of this axis indicating estimates of physical sizes within the specified regions. (From McCabe et al.49 Used by permission of the publisher.)
Map of the region surrounding the AHC and GK loci. The Xp21 region, not drawn to scale, is shown, with markers above the chromosome (horizontal black bar) and gene locations below the chromosome. The positions of the cloned MAGE (MIM 300097), AHC (MIM 300200), GK (MIM 307030), and DMD (MIM 310200) loci are accurately localized. However, the positions of the two MR phenotypes observed among patients with deletions in this region, one of which may be due to disturbance of the CNS expression of dystrophin, are given only generally. The DNA polymerase alpha gene (POLA; MIM 312040) is telomeric to this region. (This figure was drawn by W. Guo.)
Molecular genetic information on the deletions seen in patients with CGKD was crucial to characterization of GK cDNAs by three independent groups (GenBank L13943).176-178 Partial human GK cDNAs from testis, brain, and liver were identified by sequencing of random clones176 and by exon trapping,177 and the complete human hepatic GK coding region was identified by a genomic scanning method.178 The positional cloning strategies177,178 relied on cosmids that mapped to the GK critical region defined by CGKD patients. Characterization of the clones isolated by all three of these methods included appropriate mapping in patient deletion panels. Analysis of the deduced amino acid sequence for human liver GK shows a striking similarity with the prokaryotic GK sequence: 50 percent identity and 65 percent similarity with the E. coli and 47 percent identity and 63 percent similarity with the B. subtilis enzymes178,198,200 (Fig. 97-8). Human GK activity can be expressed in GK-deficient bacteria.178 In addition, the three-dimensional crystal structure for E. coli GK has been solved, and comparison with the human GK structure indicates strong conservation of amino acid residues involved in substrate interaction.178,234
Comparison of the deduced amino acid sequence for human hepatic GK with those for E. coli and B. subtilis. Amino acids in the prokaryotic sequences that are identical with (shaded) or similar to (open) those in human GK are shown in boxes. Symbols above the E. coli sequence show residues identified as interacting with the GK substrates:234 open squares indicate interactions with Mg2+; closed circles show interactions with ADP; and closed diamonds indicate interactions with glycerol. (From Guo et al.178 Used by the permission of the publisher.)
Correlation of human GK mutations with the structural features of the protein may provide insight into structure-function relationships for this protein. Four missense mutations have been described: two in symptomatic adult males with hyperglycerolemia noted incidentally (862A>G/N288D and 1313A>G/Q438R);402 one in an infant boy with neonatal asphyxia but no subsequent problems (1823T>C/M428T);402 and another in a 14-year-old male with mental retardation and no family history of mental retardation (1319A>T/D440V).403 Knowledge of the crystal structure of prokaryotic GK234 and its similarity with that of the human protein show that these mutations occur in a residue (M428) corresponding to an alanine in E. coli GK that is one member of a cluster of three amino acids interacting with ADP; near this cluster and involving absolutely conserved amino acids in E. coli, B. subtilis, and human (Q438 and D440); or in a residue (N288) that is identical in these three organisms and is near a conserved block of amino acids involved in glycerol and ADP binding. Therefore, each of these missense mutations alters an amino acid that has a defined function or is highly conserved.402
Understanding of the two additional intragenic mutations in GK relies on a knowledge of the genomic organization of this gene.181,403 One was a 61-year-old male referred because of “refractory hypertriglyceridemia” due to hyperglycerolemia, who had chronic pancreatitis in the absence of alcohol abuse, gall-stones, or hypertriglyceridemia. This man's genomic mutation involved a single base change, G>C, in the 3′ splice site of intron 6 (IVS6-1G>C), resulting in a 2-bp frameshift deletion of the first two nucleotides of exon 7, AG 553 and 554. Two brothers410 had a genomic deletion resulting in loss of the 81-bp exon 17. Despite the same degree of enzyme deficiency in the two boys, the 7-year-old brother had severe mental retardation, seizures, bone dysplasia, fractures from minor trauma and abnormal teeth, and the 3-year-old brother was clinically normal. The authors speculated that their results might indicate ascertainment bias in identification of patients with signs and symptoms that are unrelated to the GKD; a role for precipitating factors, such as metabolic or environmental stress, in revealing the predisposition to symptoms among those with GKD, as has been suggested by others398 (Bonham, personal communication); and/or possible interactions between GK mutations and other genetic polymorphisms in the individual.403
Investigation of the relationship between genotype and phenotype is required in additional patients, including those with the “classic” symptomatic, or juvenile, form of isolated GKD. If no obvious relationship can be determined between the GK genotype and phenotype, this may suggest the involvement of environmental/metabolic insult and/or modifying genes398,403 (Bonham, personal communication). As more patients are ascertained and carefully characterized at the molecular genetic level, the results may either confirm the discrete clinical classification presented above or suggest that the spectrum of phenotypes is broader than previously recognized, with the distinctions between the “classic” clinical delineations less distinct.
Additional possible loci in this region have been suggested by the phenotypes of patients with deletions, including loci for mental retardation, Oregon eye disease (OED), and HH. All patients with the CGKD characterized by AHC, GKD, and DMD have been developmentally delayed, but three with AHC and GKD without myopathy have been reported to be normal developmentally,21,34,36,417 suggesting the possibility of a mental retardation locus between GK and DMD or perhaps involving the DMD locus and affecting the brain expression of dystrophin or a related Xp21-encoded protein (Fig. 97-5).418-423 There also appears to be one or more mental retardation loci telomeric to AHC, since patients with isolated AHC and DNA deletions have been mentally retarded48,50,424 and a patient with an interstitial deletion distal to that involved in AHC had isolated mental retardation (Fig. 97-5).425 A patient with AHC, GKD, DMD, and a visible Xp21 deletion was also noted to have typical ophthalmologic features of Åland Island eye disease (AIED).39,42,43 However, since AIED426,427 maps to Xp11.4-p11.23,428 the nonallelic Xp21 locus for this phenotypically similar disorder was designated Oregon eye disease.429 This retinal abnormality is now recognized to be due to loss or malfunction of dystrophin in patients with Becker or Duchenne muscular dystrophy.430,431 An HH locus was proposed by two groups to be localized telomeric to AHC, based on deletions in patients.31,33 But, as shown for OED, if a single gene product has pleiotropic effects, then deletion mapping will indicate that these characteristics appear to map adjacently. Individuals with isolated AHC had been reported with gonadotropin deficiency and cryptorchidism,406,432 and the initial papers identifying DAX1 as the gene responsible for AHC showed that individuals with intragenic mutations in DAX1 had both AHC and HH404,405,433 (see Chap. 167). Therefore, among the phenotypes suggested by patients' deletions, to date there is clear evidence only for a distinct mental retardation (MR) locus telomeric to DAX1.
The possibility that the association between GKD and the other phenotypic features was not causal but rather coincidental was considered early in the description of this disorder, and it was hypothesized that these features might represent distinct but closely linked loci on the X chromosome.7,332 It is now quite clear that this is a contiguous gene syndrome with involvement of discrete AHC, GK, and DMD loci. The size of the genomic region involved in complex GKD is variable but can be quite large49 (Fig. 97-6), and undoubtedly additional genes that influence the patients' phenotypes will be identified in this region.
Patients with complex GKD and the juvenile form of GKD have episodic vomiting and acidemia, which responds to dietary limitation of glycerol, as noted previously. One could speculate that episodic vomiting and acidosis are associated with more significant mutations in the GK locus than those that cause the benign form of individuals with isolated GKD, but it has been suggested that those with the clinical appearance of the benign form of GKD have not been stressed metabolically.398 Additional clinical experience and molecular genetic detail will be required to resolve this point. The similarity of these episodes with those observed among patients with glycerol intolerance is intriguing, as is the possibility that disrupted compartmentation of brain GK may play a role.
A mouse model of GKD has been generated by targeted disruption of the murine Gyk gene, and all of the hemizygous mutant males die by day 3 or 4 of life with no obvious cause of death.434 The mutant males have striking hyperglycerolemia (>80-fold normal), indicating a very large flux of glycerol through glycerolipid metabolism, which is phosphorylated by Gyk for reutilization in normal animals. Elevation of plasma free fatty acid levels to approximately three times normal in mutant compared with wild type animals is consistent with a quantitatively important role for Gyk in free fatty acid reesterification, although increased free fatty acid synthesis in the Gyk knockout mice cannot be excluded as an alternative explanation. The potential involvement of these metabolic perturbations in the pathogenesis of Gyk deficiency in the mutant mice has yet to be determined. The Gyk protein shows near identity to the ATP-stimulated glucocorticoid receptor translocating promoter (ASTP), a rat liver histone-binding protein that increases the nuclear binding of the activated glucocorticoid receptor complex in the presence of ATP.180 This sequence similarity has suggested that Gyk deficiency may result not only in hyperglycerolemia and glyceroluria but also in end-organ resistance to glucocorticoid action.434 The neonatal lethality of the Gyk knockout mice has limited its utility in helping us understand the pathogenesis of symptomatic GKD.
Other Causes of Glyceroluria or Hyperglycerolemia
Glyceroluria was observed in four mentally retarded individuals among a group of 900 surveyed.1 One of these had Down syndrome, and three were reported to have cerebral palsy. Subsequently, glyceroluria was recognized in a normal female.2 Plasma glycerol concentrations were not elevated (patients, 0.06 to 0.10 millimolar; controls, 0.02 to 0.08 millimolar),1 suggesting that these patients did not have generalized GKD.5 Twenty boys with DMD were screened for glyceroluria, and one boy was positive.15 His urinary glycerol ranged from 3.2 to 10 millimolar with a mean of 7.7 millimolar, which was 40 times the upper limit of normal (≤0.2 millimolar) but was less than the concentration in the GK-deficient patient (300 millimolar) examined by the same authors and was less than the values reported for other patients (see “Diagnosis”). Serum glycerol was normal (0.15 millimolar; normal, (≤0.2 millimolar), and fibroblast GK activity was above the control range for the authors' laboratory. This patient had no evidence of MR, adrenal hypoplasia, or cytogenetic or molecular deletions.
A series of 3450 urine specimens from healthy workers, adult psychiatric patients, and children selected for metabolic screening were evaluated for glyceroluria.411 Only two were considered to have “major glyceroluria” (defined as 80 to 120 millimolar), and both of these patients had GKD.21,25 Four adults and five children had “minor glyceroluria” (defined as 2 to 10 millimolar). Three healthy workers were not investigated further; the fourth was a 33-year-old woman with developmental delay and normal blood glycerol. Two of the children were sibs, a male and a female, with an unknown, familial progressive neurodegenerative disease, mild glyceroluria, and normal blood glycerol and fibroblast GK activity. A 2-year-old boy had mild congenital myopathy and persistent low-level glyceroluria. The fourth was a female infant with infantile spasms, and the fifth was a male, but no further information was available.
Hyperglycerolemia may be seen in patients with diabetes mellitus and hyperthyroidism.5 The reported values are only severalfold higher than those in controls, and in our experience, even in patients with poorly controlled diabetes mellitus, hyperglycerolemia has not been similar to that in GKD patients. Therapeutic doses of glycerol may result in substantial hyperglycerolemia and glyceroluria,5,391 as may diagnostic doses of glycerol435 used for detection of endolymphatic hydrops in the differential diagnosis of low frequency hearing loss.393
Our experience in a metabolic screening laboratory indicates that the most common cause of glyceroluria not associated with GKD is contamination of the specimen by exogenous glycerol. Sources of contamination, especially in a “bagged” specimen collected from an infant, include glycerol-containing perineal lotions and glycerin suppositories. Other exogenous sources of glycerol include blood sampling tubes with glycerol-lubricated stoppers, detergents in the laboratory, filters used for sterilization of solutions, and moisturizing lotions used by laboratory personnel.435