Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase [beta] subunit (PHKB)
Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase [beta] subunit ( PHKB )Barbara Burwinkel1, Andrea J. Maichele1, Øystein Aagenaes2, Henk D. Bakker3, Aaron Lerner4, Yoon S. Shin5, Judith A. Strachan6 and Manfred W. Kilimann1,*
1Institut für Physiologische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany, 2Pediatric Department, Aker University Hospital, N-0514 Oslo, Norway, 3Emma Kinderziekenhuis, Universiteit van Amsterdam, NL-1105 AZ Amsterdam, The Netherlands, 4Department of Pediatrics, Carmel Medical Center, IL-34362 Haifa, Israel, 5Stoffwechselzentrum, Dr. v. Haunersches Kinderspital der LMU München, D-80337 München, Germany and 6Directorate of Biochemical Medicine, Ninewells Hospital, Dundee DD1 9SY, Scotland, UK
Received Februrary 13, 1997;Revised and Accepted April 4, 1997
Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue-specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed [beta] subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known [beta] subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of ~10%.
Deficiency of phosphorylase kinase (Phk), a regulatory protein kinase in glycogen metabolism, is responsible for one quarter of all cases of glycogen storage disease and occurs with a frequency of ~1 in 100 000 births. Phk deficiency occurs in several variants that differ in mode of inheritance (X-linked or autosomal), tissue-involvement (liver, muscle, liver and muscle, liver and kidney, heart) or clinical course (1 ,2 ). Patients affected with glycogenosis of the liver due to Phk deficiency present as infants with hepatomegaly, retardation of growth and motor development, and a characteristic rounded face (`dollface'). Hypoglycemia and elevated transaminases, triglycerides and cholesterol are often observed. Typically, patients improve with age and are often asymptomatic as adults.
The heterogeneity of Phk deficiency is thought to be primarily due to the fact that Phk is composed of four different subunits, each of which has several isoforms or splicing variants. Mutations have recently been characterized in the genes encoding the muscle and liver isoforms of the regulatory [alpha] subunit (PHKA1 and PHKA2) (2 -8 ) and the testis/liver isoform of the catalytic [gamma] subunit (PHKG2) (9 ) which explain X-linked muscle-specific, X-linked liver-specific and autosomal liver-specific Phk deficiency, respectively. Only one gene, PHKB, is known to encode the regulatory [beta] subunit. Thirty exons of this gene are expressed in all tissues investigated (liver, muscle, brain and heart) whereas three exons are subject to tissue-specific differential splicing (10 -12 ). PHKB is located on human chromosome 16q12-q13 (13 ) and was therefore a candidate for involvement in another variant of Phk deficiency, autosomal-recessive Phk deficiency affecting both liver and muscle.
In the present study, we searched the PHKB coding sequence of several patients with this form of glycogen storage disease, using a strategy based primarily on RT-PCR of RNA isolated from full blood and complemented by PCR of genomic DNA. We report here the first mutations identified in the PHKB gene. Five independent nonsense mutations, a single-base insertion, a splice-site mutation and an extensive intragenic deletion demonstrate that mutations in PHKB cause Phk deficiency of liver and muscle.
RNA was purified from deep-frozen blood samples, reverse-transcribed, amplified by PCR in several overlapping segments covering the complete coding sequence (Table 1 ), and analyzed by direct sequencing as previously described (8 ).
Patient 1 was found to be heterozygous for two PHKB mutations. A stretch of seven A residues in exon 14 is extended by an additional A resulting in a frameshift in codon 421 and translation termination at the following triplet, and a C to T transition converts glutamine 656 in exon 21 to a stop codon (Fig. 1 ). RNA and genomic DNA from the patient's parents were also analyzed by PCR and sequencing in both regions, confirming the mutations and demonstrating that the frameshift mutation had been inherited from the father and the nonsense mutation from the mother.
Patient 2 was found to be heterozygous for three different mutations: two nonsense mutations in codons Y418 (exon 14) and E975 (exon 31) and a Y974H missense mutation (Fig. 1 ). Surprisingly, RT-PCR analysis of RNA from both parents did not yield significant sequence signals for any of these mutations. As translation-terminating mutations often result in reduced abundance of the mRNA (ref. 2 and references therein), we suspected that this might also be the case for both mutant alleles in this family, so that they would be detectable together in the patient but missed against the background of the higher levels of mRNA from the normal alleles in the parents. We therefore amplified exons 14 and 31 from genomic DNA of both parents and could indeed identify, with signal intensities similar to the normal alleles, the Y418ter mutation in the father and a Y974H plus E975ter double mutation in the mother.
The ATTA deletion near the end of intron 20 abolishes an AsnI restriction site. To investigate whether it is polymorphic, the genomic DNA of additional, unrelated individuals was analyzed by PCR of the [beta]i22/i23 interval (sequence at the bottom of Fig. 2 ), AsnI digestion, and agarose gel electrophoresis of the resultant DNA fragments (not shown). Of 31 chromosomes analyzed, 20 (65%) were positive for ATTA/AsnI and 11 (35%) were negative, identifying this structural feature as the first polymorphic marker in the PHKB gene at 16q12-q13.
Patient 5 was expected to have a homozygous mutation because his parents are consanguineous. Indeed, a homogeneous amplification product of reduced length was obtained by RT-PCR with primers [beta]m16/m29 (Fig. 3 a). Its sequence revealed the absence of exon 8 (not shown), resulting in a frameshift and truncation of the reading frame after only 19% of its normal length. An unaffected brother and two unrelated controls yielded normal-sized RT-PCR products whereas products of both sizes could be amplified from RNA of both parents, indicating heterozygosity (Fig. 3 a). We then tried to amplify exon 8 and its immediate vicinity from the patient's genomic DNA with primers [beta]m33/m34 but obtained no product, in contrast to DNA from eight control individuals (Fig. 3 b, additional controls not shown). To obtain more information on the extent and nature of the deletion of exon 8 in the genomic DNA of patient 5, we then sequenced the 6 knt genomic HindIII fragment containing exon 8 (subclone [beta]2-H1; ref. 10 ), finding that exon 8 lies within a cluster of retroposons (Fig. 3 c). There are two Alu elements, 1 and 2 knt downstream of exon 8, and an inverted Alu half-element lies immediately upstream of the exon. This constellation is flanked by two LINE-1 elements, both in orientation opposite to that of PHKB and both apparently extending beyond the borders of subclone [beta]2-H1. The upstream LINE-1 begins immediately adjacent to the Alu half-element and is cut off at nt 742 of the LINE-1 reference sequence by the HindIII site, beyond which it may continue for up to 5500 nt. The downstream LINE-1 sets in at the right-hand HindIII site with nt 1882 and is truncated at nt 3653 of the LINE-1 reference sequence. Thus, exon 8 is surrounded by long stretches of potentially recombinogenic repetitive DNA elements that might be responsible for the deletion. Next, we investigated whether this constellation of repetitive elements found in clone [beta]2-H1 is indeed representative for the normal gene structure. Two pairs of primers were designed to amplify sequence intervals extending from the unique sequence region into the upstream or downstream LINE-1 elements ([beta]i34/m34 and [beta]i32/i33, respectively, Fig. 3 c). PCR products of expected sizes and terminal sequences were obtained from all six control individuals but not from patient 5, confirming that clone [beta]2-H1 represents the normal structure surrounding exon 8 of the PHKB gene and that this structure is disrupted in patient 5. Several other sequence intervals were probed by PCR (Fig. 3 c), and all yielded specific products (confirmed by sequencing) from control individuals but not from patient 5. Hence, the deletion in patient 5 extends over at least 6 knt between the positions of primers [beta]i41 and [beta]i43.ab
The five independent nonsense mutations, one single-base insertion, one splice-site mutation and one large intragenic deletion identified here demonstrate that mutations in the PHKB gene cause autosomal-recessive glycogenosis due to Phk deficiency of both liver and muscle. Patients 2, 4 and 5 are exceptionally well-characterized published cases where Phk deficiency had been determined in muscle biopsies as well as in liver or erythrocytes (see Materials and Methods). PHKB therefore was our first candidate gene for the mutation search which proved successful in all three cases. In normal diagnostic practice, however, Phk activity measurements in muscle and liver are rarely carried out. Symptoms of liver involvement predominate, and the presence or absence of clinical muscle symptoms is inconclusive (9 ) so that muscle is rarely biopsied. In fact, even liver biopsies are often avoided and activity determinations only performed on erythrocytes. Family history is also seldom so suggestive of autosomal-recessive inheritance as in cases 2 and 5 (Materials and Methods). Therefore, it is often difficult to predict whether a case of liver glycogenosis due to Phk deficiency is of the X-linked liver-specific (mutations in PHKA2), the autosomal liver-specific (mutations in PHKG2) or of the autosomal liver-and-muscle type (mutations in PHKB). Patients 1 and 3 are examples who had no muscle symptoms and whose muscle was therefore not analyzed. In patient 1, who is male, we first searched the X-chromosomal PHKA2 gene and then PHKG2, candidate genes in which mutations do not affect Phk activity in muscle, before finally finding the mutations in PHKB. As patient 3 is female, autosomal inheritance was suspected from the beginning but again, PHKG2 was analyzed first. Liver glycogenosis due to Phk deficiency is generally benign and tends to improve clinically with increasing age. This is also observed for the older patients with mutations in PHKB (see Methods). Indeed, it appears that cases with PHKB mutations are even more mildly affected than PHKA2 and PHKG2 cases, with relatively high residual Phk activities around 10% in affected tissues, and transaminases and triglycerides not or only moderately enhanced. Impairment of muscle function by this type of Phk deficiency may be so mild that it is never noted (patients 1 and 3). However, it remains to be seen whether more significant muscle symptoms will develop in the long run, as some patients with muscle-specific Phk deficiency have developed complaints only as adults. Also in glycogenosis type III (debranching enzyme deficiency) it is observed that `although the enzyme defect is expressed in both liver and muscle in most patients, clinical myopathy is not common and often manifests in adult life, long after the liver symptoms have remitted' (14 ).
There are five known genes for Phk subunits (not counting the subunit [delta] which is calmodulin), and the PHKB gene is the fourth in which mutations have been identified. The fifth, PHKG1, encodes the muscle isoform of the catalytic [gamma] subunit and is a candidate for autosomal muscle-specific Phk deficiency. Thus, the genes associated with most, and the most frequent, forms of Phk deficiency are now known. However, there remain unresolved questions in the molecular genetics of Phk. In particular, we still do not understand the molecular basis of the very rare and clinically most severe forms, hepato-renal (15 ,16 ) and heart-specific (17 -19 ) Phk deficiency. The results of the present study emphasize this. PHKB is the only known gene of the [beta] subunit, and all mutations described here are in exons that are believed to be expressed in all tissues. These mutations therefore met our expectations insofar as they cause Phk deficiency in liver as well as in muscle. However, it is unclear why they do not also affect the kidney or the heart. It is also striking that although all these mutations are severe translation-terminating mutations that lead to truncated protein products unlikely to have residual function, patients mostly have notable residual Phk activities around 10% in the affected tissues, liver and muscle, and even higher values in erythrocytes, leukocytes or fibroblasts. In contrast, a patient with a mutation terminating the PHKA1 gene product after 90% of its normal length (3 ) had only 0.3% residual activity in muscle, in spite of the existence of a second isoform gene for the [alpha] subunit. This may be explained by the existence of another, yet unidentified [beta] subunit gene. It is also possible that the [beta] subunit can be replaced in the holoenzyme by the partially homologous [alpha] subunit but not vice versa, or that a partially stable and active complex can be formed without [beta] but not without [alpha]. Indeed, partial denaturation of Phk with LiBr, or partial proteolysis, is known to produce an [alpha][gamma][delta] complex that is catalytically active (20 ,21 ).
Purification of RNA and genomic DNA from deep-frozen blood samples was performed according to conventional procedures. RT-PCR and direct cycle-sequencing are described in detail in ref. 8 . All mutations were confirmed from two or more independent RNA or DNA preparations. The complete PHKB coding sequence was analyzed in patients 1, 2, 4 and 5 , and ~50% of the coding sequence was analyzed in patient 3, without finding other sequence abnormalities.
Patient 1 (FB.M, male), the only child of unrelated, healthy German parents, was admitted at the age of 22 months because of an extended abdomen due to hepatomegaly. Transaminases and triglycerides were slightly elevated. Phk activity in erythrocytes was markedly decreased (12% residual activity) while the parents' erythrocyte Phk activities were in the heterozygote range (mother: 56%; father: 40% of normal). Glycogen in erythrocytes was high (10 mg/dL; normal range: 0-10 mg/dL) and the phosphorylase a/a+b ratio in leukocytes very low (0.03; normal range 0.4-0.5). At present (age 4 years), the child presents with body height at the 10th and weight at the 50th percentile, hepatomegaly, and a tendency to develop hypoglycemic symptoms after several hours of fasting or physical activity. There are no clinical indications of muscle involvement. Also the mother reports that she has hypoglycemic symptoms upon physical exercise that are countered by carbohydrate intake.
Investigations of patients 2, 4 and 5 have been published before, demonstrating Phk deficiency in liver or erythrocytes as well as in muscle and suggesting an autosomal-recessive mode of inheritance in cases 2 and 5. Patient 2 (LH.O, male; refs 22 ,23 ) and his sister (also affected) are the children of unaffected, unrelated Norwegian parents. They came to medical attention as infants because of hepatomegaly; glucagon response by both was normal. Residual Phk activities were 18% of normal in hemolysates of both, 5% in liver of the sister (but the phosphorylase a/a+b ratio, 0.5, was surprisingly normal), and 0-13% (depending on pH) in muscle of patient 2 (a/a+b ratio: 0.07). Today, at age ~25, both individuals are fully capable of everyday physical activities, but tend to develop hypoglycemic symptoms upon activity or fasting which are ameliorated by carbohydrate intake. Hepatomegaly has receded, clinical muscle symptoms have never been noted.
Patient 3 (PB.A, female), now 6 years old, is the daughter of healthy Dutch parents and has one brother and one sister, both unaffected. Although she is apparently homozygous for her splice-site mutation, the parents are reportedly unrelated but originate from the same geographical region. She came to medical attention because of an extended abdomen due to hepatomegaly but is free of any symptoms of hypoglycemia or muscle involvement. Transaminases were found slightly enhanced on occasion but mostly normal, triglycerides and cholesterol were normal. Phk activity was undetectable in liver and 12% of controls in erythrocytes. Remarkably, a nearly normal phosphorylase a/a+b ratio, 0.43, was determined in erythrocytes.
The parents of patient 4 (KM.D, male, British; refs 24 ,25 ) are healthy and unrelated and there is one healthy half-brother. The patient, now 15 years old, was referred at age 5 years because of abdominal extension (noted since early infancy) and muscle weakness. He presented with height on the 25th percentile, a doll-like face, thin extremities, hepatomegaly, and reduced muscle power and bulk. He was never found to be hypoglycemic, and the glucagon response was normal. Laboratory findings included normal creatine kinase, slightly raised aspartate aminotransferase, slight fibrosis and enhanced glycogen (8.8 g%; normal <5 g%) in a liver biopsy, and reduced Phk activity both in erythrocytes (13% of normal) and in muscle (9%). Muscle glycogen was high (1.35 g%; normal, 0.34) and the a/a+b ratio very low (0.01).
Patient 5 (MH.H, male, Israeli-Arab; refs 26 ,27 ) has two healthy brothers and two affected sisters. His healthy parents are distantly related. A protuberant abdomen had been noted since 18 months of age. Upon admission at age 4 years, severe hepatomegaly, a doll-face, mild generalized muscular hypotonia, height below the third percentile and weight in the 20th percentile were observed. There was no history of hypoglycemic symptoms, and blood glucose levels and a glucagon test were normal. In a liver biopsy, strongly elevated glycogen (17 g%; controls, 1-5 g%) undetectable phosphorylase a, and Phk activity reduced to 20% of normal were found. A muscle biopsy also demonstrated glycogenosis (1.9 g%; controls, 0.3-1 g%) and Phk deficiency (25% residual activity). At present, neither the patient (age 21) nor his elder sisters display any notable symptoms (no hepatomegaly, normal height, no impairment of everyday physical activity).
We thank the patients and their families for their support. Many thanks for mediating access to patients are also due to Drs G. Besley, M. Faed, V. Marrian and D. Walsh (Manchester/Perth/Dundee), Dr O. Søvik (Bergen) and Dr S. Moses (Beersheva). Subclone [beta]2-H1 was prepared by Dr A. Wüllrich-Schmoll. This work was funded by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
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*To whom correspondence should be addressed. Tel: +49 234 700 7927; Fax +49 234 7094 193; Email: manfred.kilimann{at}rz.ruhr-uni-bochum.de
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