J. Inher. Metab. Dis. 15 (1992) 335 338 9 SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
Short Communication
Biochemical and DNA Markers of X-Linked Hypophosphataemic Rickets: A Study of Sporadic Cases E. PRONICKA, E. POPOWSKA, E. ROWI/qSKA, D. PIEKUTOWSKA, M. OGLECKA and M. KRAJEWSKA-WALASEK Department of Metabolic Diseases and Department of Genetics, Child Health Centre, Al Ozieci Polskich 20, Warsaw, Poland In 1958 Winters and co-workers, having studied a number of large kindreds from North Carolina, demonstrated definitively that familial hypophosphataemic rickets (FHR: McKusick 307800) is inherited as an X-linked dominant trait and that hypophosphataemia is the most reliable marker of the presence of the mutant gene. Soon after that Greenberg and co-workers (1960) published the rules determining blood serum phosphate and interpreting the findings for purposes of genetic counselling. The two, now classic, studies have become the basic reference works in the field of FHR studies. In recent years the gene responsible for FHR has been localized to the Xp22.321.3 band and polymorphic genomic flanking probes linked to it have been produced (Read et al 1986; Machler et al 1986). This has made possible the use of the DNA marker restriction fragment length polymorphisms in family studies of hypophophataemic rickets. In available literature the data on the incidence of sporadic cases of hypophosphataemic rickets with the same phenotype as the condition with proven X-linked dominant inheritance are incomplete and often inconsistent. In 1964 Burnett and co-workers roughly estimated the frequency of a fresh mutation of the FHR gene at 6.4 x 10 - 6 and 5.3 • 10-6 in males and females, respectively. According to our own observations, the number of sporadic cases of FHR among Polish children is high. The aim of the study was to elucidate the origin of sporadic cases using the methods of DNA analysis. MATERIAL AND METHODS The Department of Metabolic Diseases, Child Health Centre, Warsaw, serves as a national consultation unit, including the diagnosis and treatment of rickets. Since the foundation in 1977, FHR has been detected in 92 children and 23 parents. In children the diagnosis was made from the commonly recognized criteria, i.e. the onset of the symptoms and signs as the child starts walking, hypophosphataemia and 335
336
Pronicka et al.
hyperphosphaturia, growth retardation, rickets-like radiological changes in the skeleton, comparatively low 1,25-dihydroxy-vitamin Da levels and resistance to treatment with vitamin Da and its derivatives. The parents, siblings and, when required, also grandparents and distant relatives of all affected children, in both familial and sporadic cases, were evaluated for post-rickets skeletal deformities, short stature and hypophosphataemia. Blood for phosphate measurements was usually collected a number of times in the fasting state or 4 hours after a meal. Values deviating by - 2 S D from the normal range for age and sex on the curves of Greenberg et al (1960) were considered as hypophosphataemia. The chromosomal DNA of the patients and unaffected family members was extracted from leukocytes isolated from 10 ml full blood, digested with the restriction enzymes Taq I and Pst I and hybridized with the probes 99.61 and D2. In selected cases hybridization with the probes XJ2.3 and 754 was also carried out after digestion with the enzymes Taq I and Pst I, respectively. RESULTS Frequency of FHR: Over a period of 15 years FHR was diagnosed in 115 individuals from 85 families including 92 children aged 0-18 years (33 boys and 59 girls). The children were referred mostly at the ages of 3 to 7 years; on average there were 8.7 new cases of FHR annually. On the assumption that our material constitutes the majority of FHR cases in Poland, the frequency of the condition was calculated. The number of probands born in the years !982-1985 was compared with the total number of children aged 2-6 years in the general population in 1988, the figures being 22 and 2736000 respectively, and the FHR frequency thus calculated as 8 • 10 -6 (1 : 124000). Frequency of sporadic cases: In only 23 probands' families studied was FHR diagnosed in the parents (5 fathers and 18 mothers). In the remaining 62 probands neither rickets nor hypophosphataemia was detected in any of the parents. FHR was sporadic in those families (73%). In the group with familial occurrence of the condition only five affected parents themselves had an affected parent (the proband's grandparent), the remaining 18 demonstrating FHR as the first in their families (78%). Similarly, in the previous generation only two of the five affected grandparents reported the condition in one of their parents (the proband's great-grandparent). The size of sibships was analysed in sporadic and familial cases of hypophosphataemic rickets. In the identified FHR families the number of siblings tallies with the figures for the Polish population, i.e. three or more children in 16.0% and 18.6% of the families respectively. In sporadic cases, however, the distribution of sibship size is strikingly different, with an evident shift towards big families. In this group there were three or more children in 49.1% of the families. DNA analyses in F H R families and sporadic cases: DNA analyses using four polymorphic genomic probes have been done so far in 43 families, including 23 known families with FHR and 20 families with sporadic cases. J. Inher. Metab. Dis. 15 (1992)
X-Linked Hypophosphataemic Rickets
337
In the identified F H R families the probes used allow us to recognize in all the subjects the R F L P allele set linked with the condition. The marker R F L P haplotype was always identified in the affected sibling of the proband (6 siblings examined) but absent in the unaffected sibling(s) who inherited the other set of alleles (10 children examined). The segregation of parental R F L P alleles was also analysed in the siblings of children with sporadic hypophosphataemic rickets, although the marker haplotype could not be determined. So far 20 families having more than one child have been examined. In contrast to the cases of familial occurrence of the condition, the unaffected sibling(s) of the patient either shared with him the same set of alleles or inherited the other, alternative, set of alleles from the mother. When the same set of alleles was identified in the affected child and at least one unaffected sibling it was considered as confirming that hypophosphataemic rickets in that family was a fresh mutation. The finding was observed in 7 cases. In 6 cases the R F L P alleles in normal sibling(s) and the affected child differed. Considering a random segregation of alleles, the difference could not be said to exclude a fresh mutation. The data of the remaining 7 cases are still non-conclusive.
Frequency of fresh mutations in the F H R gene." The frequency of new mutations was calculated by an indirect method assuming after Stevenson and Kerr (1967) that fitness was 0.4 while the frequency of the disease was estimated by us at 8 • 10 -6. Thus calculated frequency of mutations in the F H R gene was 3.2 • 10 -6. The frequency of mutations was also assessed by a direct method comparing the number of sporadic cases of hypophosphataemic rickets in children aged 2-6 years in 1988 and the entire population in this age group. The frequency obtained was similar, i.e. 4 • 10-6; 0.7 x 10 -6 for boys and 3.3 x 10 -6 for girls.
D I S C U S S I O N AND C O N C L U S I O N S Since the publication of the works by Winters, Burnett and Greenberg in the late 1950s and early 1960s there have been few studies dealing with the genetic aspects of FHR. The frequency of FHR in the population is not known precisely, but it is generally thought to be a frequent disease. For sporadic cases the figures vary and are incomplete (Stevenson and Kerr 1967; Burnett et al 1964; Briand-Guillemot et al 1972; Rasmussen and Tenenhouse 1989). We have estimated the frequency of FHR in the Polish population at 8 x 10 -6 and the new mutation rate at 4 • 10 -6. The figures could be underestimated since some F H R cases might have remained undiagnosed or might have escaped referral to our centre. A sporadic emergence of a phenotype characteristic of F H R may be accounted for by new mutations as well as a different pattern of inheritance, an incomplete dominance of the F H R gene, and, finally, though this is least likely, a phenocopy. In our opinion our findings seem to confirm that the vast majority of sporadic cases of hypophosphataemic rickets results from fresh mutations. The following suggest such a line of interpretation: J. Inher. Metab. Dis. 15 (1992)
338 (1)
(2) (3)
Pronicka et al.
Over 70% of the affected parents of our F H R patients were previously considered to be sporadic cases themselves. It was the birth of an affected child that revealed the familial nature of the condition. In large sibships at higher risk of a new mutation, sporadic cases were significantly more frequent. The distribution of paternal alleles of R F L P s known to be linked to the F H R gene in healthy siblings of patients with sporadic hypophosphataemic rickets was random. In over half of the cases the affected child shared the allele set with one of the healthy siblings. This excludes the hypothesis of an incomplete dominance of the F H R gene and confirms the likelihood of a new mutation.
Further observations and studies are required for the final elucidation of the origin of sporadic cases of hypophosphataemic rickets. For the purposes of genetic counselling, after hypophosphataemia has been excluded in the parents, sporadic cases of hypophosphataemic rickets should be considered as a new mutation in the F H R gene.
REFERENCES Briand-Guillemot ML, Raverdy E, Balsan S, Rey J, Fr6zal J (1972) Etude critique de l'hypophosphatemie pour l'etude genetique du rachitisme vitamino-resistant hypophosphatemique familial. Arch Franc Ped 29: 1059. Burnett Ch, Dent CE, Harper C, Warland BJ (1964) Vitamin D-resistant rickets: Analysis of 24 pedigrees with hereditary and sporadic cases. Am J Med 36: 222. Greenberg BG, Winters RW, Graham JB (1960) The normal range of serum inorganic phosphorus and its utility as a discriminant in the diagnosis of congenital hypophosphatemia. J Clin Endocrinol 20: 364. Machler M, Frey D, Gal Aet al (1986) X-linked dominant hypophosphatemia is closely linked to DNA markers DXS41 and DS43 at Xp22. Hum Genet 73: 271. Rasmussen H, Tenenhouse HB (1989) Hypophosphatemias. In Scriver CR, Beaudet AL, Sly WS, Valle D, eds. Metabolic Basis of Inherited Disease. New York: McGraw-Hill, 2581. Read AP, Thakker RV, Davies KE et al (1986) Mapping of human X-linked hypophosphatemic rickets by multilocus linkage analysis. Hum Genet 73: 267. Stevenson AC, Kerr CB (1967) On the distributions of frequencies of mutation to genes determining harmful traits in man. Mutation Res 4: 339. Winters RW, Graham JB, Williams TF, McFalk VW, Burnett CH (1958) A genetic study of familial hypophosphatemia and vitamin D-resistant rickets with a review of the literature. Medicine (Baltimore) 37: 97-142.
J. Inher. Metab. Dis. 15 (1992)
Short Communication
Biochemical and DNA Markers of X-Linked Hypophosphataemic Rickets: A Study of Sporadic Cases E. PRONICKA, E. POPOWSKA, E. ROWI/qSKA, D. PIEKUTOWSKA, M. OGLECKA and M. KRAJEWSKA-WALASEK Department of Metabolic Diseases and Department of Genetics, Child Health Centre, Al Ozieci Polskich 20, Warsaw, Poland In 1958 Winters and co-workers, having studied a number of large kindreds from North Carolina, demonstrated definitively that familial hypophosphataemic rickets (FHR: McKusick 307800) is inherited as an X-linked dominant trait and that hypophosphataemia is the most reliable marker of the presence of the mutant gene. Soon after that Greenberg and co-workers (1960) published the rules determining blood serum phosphate and interpreting the findings for purposes of genetic counselling. The two, now classic, studies have become the basic reference works in the field of FHR studies. In recent years the gene responsible for FHR has been localized to the Xp22.321.3 band and polymorphic genomic flanking probes linked to it have been produced (Read et al 1986; Machler et al 1986). This has made possible the use of the DNA marker restriction fragment length polymorphisms in family studies of hypophophataemic rickets. In available literature the data on the incidence of sporadic cases of hypophosphataemic rickets with the same phenotype as the condition with proven X-linked dominant inheritance are incomplete and often inconsistent. In 1964 Burnett and co-workers roughly estimated the frequency of a fresh mutation of the FHR gene at 6.4 x 10 - 6 and 5.3 • 10-6 in males and females, respectively. According to our own observations, the number of sporadic cases of FHR among Polish children is high. The aim of the study was to elucidate the origin of sporadic cases using the methods of DNA analysis. MATERIAL AND METHODS The Department of Metabolic Diseases, Child Health Centre, Warsaw, serves as a national consultation unit, including the diagnosis and treatment of rickets. Since the foundation in 1977, FHR has been detected in 92 children and 23 parents. In children the diagnosis was made from the commonly recognized criteria, i.e. the onset of the symptoms and signs as the child starts walking, hypophosphataemia and 335
336
Pronicka et al.
hyperphosphaturia, growth retardation, rickets-like radiological changes in the skeleton, comparatively low 1,25-dihydroxy-vitamin Da levels and resistance to treatment with vitamin Da and its derivatives. The parents, siblings and, when required, also grandparents and distant relatives of all affected children, in both familial and sporadic cases, were evaluated for post-rickets skeletal deformities, short stature and hypophosphataemia. Blood for phosphate measurements was usually collected a number of times in the fasting state or 4 hours after a meal. Values deviating by - 2 S D from the normal range for age and sex on the curves of Greenberg et al (1960) were considered as hypophosphataemia. The chromosomal DNA of the patients and unaffected family members was extracted from leukocytes isolated from 10 ml full blood, digested with the restriction enzymes Taq I and Pst I and hybridized with the probes 99.61 and D2. In selected cases hybridization with the probes XJ2.3 and 754 was also carried out after digestion with the enzymes Taq I and Pst I, respectively. RESULTS Frequency of FHR: Over a period of 15 years FHR was diagnosed in 115 individuals from 85 families including 92 children aged 0-18 years (33 boys and 59 girls). The children were referred mostly at the ages of 3 to 7 years; on average there were 8.7 new cases of FHR annually. On the assumption that our material constitutes the majority of FHR cases in Poland, the frequency of the condition was calculated. The number of probands born in the years !982-1985 was compared with the total number of children aged 2-6 years in the general population in 1988, the figures being 22 and 2736000 respectively, and the FHR frequency thus calculated as 8 • 10 -6 (1 : 124000). Frequency of sporadic cases: In only 23 probands' families studied was FHR diagnosed in the parents (5 fathers and 18 mothers). In the remaining 62 probands neither rickets nor hypophosphataemia was detected in any of the parents. FHR was sporadic in those families (73%). In the group with familial occurrence of the condition only five affected parents themselves had an affected parent (the proband's grandparent), the remaining 18 demonstrating FHR as the first in their families (78%). Similarly, in the previous generation only two of the five affected grandparents reported the condition in one of their parents (the proband's great-grandparent). The size of sibships was analysed in sporadic and familial cases of hypophosphataemic rickets. In the identified FHR families the number of siblings tallies with the figures for the Polish population, i.e. three or more children in 16.0% and 18.6% of the families respectively. In sporadic cases, however, the distribution of sibship size is strikingly different, with an evident shift towards big families. In this group there were three or more children in 49.1% of the families. DNA analyses in F H R families and sporadic cases: DNA analyses using four polymorphic genomic probes have been done so far in 43 families, including 23 known families with FHR and 20 families with sporadic cases. J. Inher. Metab. Dis. 15 (1992)
X-Linked Hypophosphataemic Rickets
337
In the identified F H R families the probes used allow us to recognize in all the subjects the R F L P allele set linked with the condition. The marker R F L P haplotype was always identified in the affected sibling of the proband (6 siblings examined) but absent in the unaffected sibling(s) who inherited the other set of alleles (10 children examined). The segregation of parental R F L P alleles was also analysed in the siblings of children with sporadic hypophosphataemic rickets, although the marker haplotype could not be determined. So far 20 families having more than one child have been examined. In contrast to the cases of familial occurrence of the condition, the unaffected sibling(s) of the patient either shared with him the same set of alleles or inherited the other, alternative, set of alleles from the mother. When the same set of alleles was identified in the affected child and at least one unaffected sibling it was considered as confirming that hypophosphataemic rickets in that family was a fresh mutation. The finding was observed in 7 cases. In 6 cases the R F L P alleles in normal sibling(s) and the affected child differed. Considering a random segregation of alleles, the difference could not be said to exclude a fresh mutation. The data of the remaining 7 cases are still non-conclusive.
Frequency of fresh mutations in the F H R gene." The frequency of new mutations was calculated by an indirect method assuming after Stevenson and Kerr (1967) that fitness was 0.4 while the frequency of the disease was estimated by us at 8 • 10 -6. Thus calculated frequency of mutations in the F H R gene was 3.2 • 10 -6. The frequency of mutations was also assessed by a direct method comparing the number of sporadic cases of hypophosphataemic rickets in children aged 2-6 years in 1988 and the entire population in this age group. The frequency obtained was similar, i.e. 4 • 10-6; 0.7 x 10 -6 for boys and 3.3 x 10 -6 for girls.
D I S C U S S I O N AND C O N C L U S I O N S Since the publication of the works by Winters, Burnett and Greenberg in the late 1950s and early 1960s there have been few studies dealing with the genetic aspects of FHR. The frequency of FHR in the population is not known precisely, but it is generally thought to be a frequent disease. For sporadic cases the figures vary and are incomplete (Stevenson and Kerr 1967; Burnett et al 1964; Briand-Guillemot et al 1972; Rasmussen and Tenenhouse 1989). We have estimated the frequency of FHR in the Polish population at 8 x 10 -6 and the new mutation rate at 4 • 10 -6. The figures could be underestimated since some F H R cases might have remained undiagnosed or might have escaped referral to our centre. A sporadic emergence of a phenotype characteristic of F H R may be accounted for by new mutations as well as a different pattern of inheritance, an incomplete dominance of the F H R gene, and, finally, though this is least likely, a phenocopy. In our opinion our findings seem to confirm that the vast majority of sporadic cases of hypophosphataemic rickets results from fresh mutations. The following suggest such a line of interpretation: J. Inher. Metab. Dis. 15 (1992)
338 (1)
(2) (3)
Pronicka et al.
Over 70% of the affected parents of our F H R patients were previously considered to be sporadic cases themselves. It was the birth of an affected child that revealed the familial nature of the condition. In large sibships at higher risk of a new mutation, sporadic cases were significantly more frequent. The distribution of paternal alleles of R F L P s known to be linked to the F H R gene in healthy siblings of patients with sporadic hypophosphataemic rickets was random. In over half of the cases the affected child shared the allele set with one of the healthy siblings. This excludes the hypothesis of an incomplete dominance of the F H R gene and confirms the likelihood of a new mutation.
Further observations and studies are required for the final elucidation of the origin of sporadic cases of hypophosphataemic rickets. For the purposes of genetic counselling, after hypophosphataemia has been excluded in the parents, sporadic cases of hypophosphataemic rickets should be considered as a new mutation in the F H R gene.
REFERENCES Briand-Guillemot ML, Raverdy E, Balsan S, Rey J, Fr6zal J (1972) Etude critique de l'hypophosphatemie pour l'etude genetique du rachitisme vitamino-resistant hypophosphatemique familial. Arch Franc Ped 29: 1059. Burnett Ch, Dent CE, Harper C, Warland BJ (1964) Vitamin D-resistant rickets: Analysis of 24 pedigrees with hereditary and sporadic cases. Am J Med 36: 222. Greenberg BG, Winters RW, Graham JB (1960) The normal range of serum inorganic phosphorus and its utility as a discriminant in the diagnosis of congenital hypophosphatemia. J Clin Endocrinol 20: 364. Machler M, Frey D, Gal Aet al (1986) X-linked dominant hypophosphatemia is closely linked to DNA markers DXS41 and DS43 at Xp22. Hum Genet 73: 271. Rasmussen H, Tenenhouse HB (1989) Hypophosphatemias. In Scriver CR, Beaudet AL, Sly WS, Valle D, eds. Metabolic Basis of Inherited Disease. New York: McGraw-Hill, 2581. Read AP, Thakker RV, Davies KE et al (1986) Mapping of human X-linked hypophosphatemic rickets by multilocus linkage analysis. Hum Genet 73: 267. Stevenson AC, Kerr CB (1967) On the distributions of frequencies of mutation to genes determining harmful traits in man. Mutation Res 4: 339. Winters RW, Graham JB, Williams TF, McFalk VW, Burnett CH (1958) A genetic study of familial hypophosphatemia and vitamin D-resistant rickets with a review of the literature. Medicine (Baltimore) 37: 97-142.
J. Inher. Metab. Dis. 15 (1992)
