0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society
Vol. 86, No. 3 Printed in U.S.A.
Maternal Isodisomy for Chromosome 2p Causing Severe Congenital Hypothyroidism BERT BAKKER*, HENNIE BIKKER*, RAOUL C. M. HENNEKAM, ¨ TTE G. J. SCHIPPER, THOMAS VULSMA, ED J. P. LOMMEN, MARIE JAN J. M. DE VIJLDER
AND
Academic Medical Center, University of Amsterdam, Emma Children’s Hospital AMC, Division of Pediatric Endocrinology (B.B., H.B., T.V., J.J.M.d.V.), Department of Clinical Genetics, Institute for Clinical Genetics (M.S., R.H.), 1100 DE Amsterdam, The Netherlands; and St. Joseph Hospital, Department of Pediatrics (E.J.P.L.), 5500 MB Veldhoven, The Netherlands ABSTRACT Severe congenital hypothyroidism (CH) due to a total iodide organification defect (TIOD) is usually due to mutations in the thyroid peroxidase (TPO) gene located at chromosome 2p25. A homozygous deletion [⌬T2512 (codon 808)] in exon 14 was identified in a patient with classical TIOD. The transmission pattern of the TPO gene in this family was anomalous; the mother was heterozygous for the deletion; and the mutation was absent in the father. Polymorphic short tandem repeat (STR) markers confirmed paternity and demonstrated on chromosome 2 that the propositus was homozygous for most markers on chromosome 2p and that these were identical to one of the maternal
T
HE TOTAL iodide organification defect (TIOD) is a frequent hereditary cause of severe congenital hypothyroidism (CH) in The Netherlands, present in 1 out of every 60,000 newborns (1). The clinico-pathologic presentation is with extremely low plasma FT4 and T4 levels, high plasma TSH and thyroglobulin concentrations, elevated 123-I uptake in the thyroid gland, and an immediate and complete release of intrathyroidal 123-I after sodium perchlorate administration (2). TIOD is a recessively inherited disorder caused by mutations in the thyroid peroxidase (TPO) gene on the short arm of chromosome 2 at p25 (3–5). TPO is a thyroid-specific enzyme playing a key role in thyroid hormone biosynthesis. It catalyzes iodination of tyrosine residues and coupling of iodotyrosine residues at the apical border of the thyroid follicular cell (6). In this study, we report on a patient with a TIOD in which homozygosity for a TPO mutation was caused by partial maternal isodisomy for chromosome 2p. Materials and Methods Mutation analysis Genomic DNA was extracted from peripheral blood lymphocytes by standard methods, using QIAGEN G100 Genomic tips (QIAGEN, Valencia, CA). The coding sequence and intron/exon boundaries of the TPO gene of Received April 8, 2000. Revised October 25, 2000. Accepted November 1, 2000. Address all correspondence and requests for reprints to: Hennie Bikker, Ph.D., Academic Medical Center, University of Amsterdam, Emma Children’s Hospital AMC, Division of Pediatric Endocrinology, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: h.bikker@amc. uva.nl or
[email protected]. * Both authors contributed equally to this work.
2p homologs. A normal karyotype was found in the propositus, his parents and sister. We conclude that the homozygosity in the patient is due to partial maternal isodisomy of the short arm of chromosome 2, carrying a defective TPO gene. The patient, born small for gestational age, develops and grows well and appears healthy (while being treated with thyroxine) and has a normal phenotype except for a unilateral preauricular skin tag. This shows that partial maternal isodisomy for chromosome 2p (2pter - 2p12) is compatible with a minimal influence on normal development. (J Clin Endocrinol Metab 86: 1164 –1168, 2001)
the proband was studied using restriction enzyme digestion and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified genomic DNA (5, 7). Exons with aberrant DGGE patterns were sequenced using ABI Prism BigDye primer cycle sequencing chemistry (Perkin-Elmer Corp. PE Applied Biosystems, Foster City, CA) on an ABI 377 DNA sequencer.
Locus specific microsatellite marker analysis Genotypic analysis with polymorphic STR markers was used to validate paternity (Geneprint Powerplex 1.2 system, Promega Corp., Madison, WI) on an ABI Prism 310 Genetic Analyzer. For genotyping of chromosome 2, we used the following markers: D2S319, D2S162, D2S149, D2S165, D2S170, D2S367, D2S2259, D2S391, D2S123, D2S337, D2S2368, D2S101, D2S286, D2S2333, D2S274, D2S2229, D2S2386, D2S160, D2S347, D2S112, D2S142, D2S335, D2S364, D2S202, D2S117, D2S325, D2S2382 and D2S126, see Fig. 2. Two additional STR markers (D2S2216 and D2S113) were not informative. The sequences of the oligonucleotide primers for the microsatellite markers were obtained from the Genome Database (http://www.gdb.org). Forward primers were Cy5-labeled. PCR reactions contained 30 ng of genomic DNA, 150 m of each dATP, dCTP, dGTP, dTTP, 0.32 m of each primer and 0.3 U Taq polymerase (SuperTaq, HT Biotechnology Ltd.) in 15 L of buffer consisting of 10 mm Tris-HCl pH 9.0, 1.5 mm MgCl2, 50 mm KCl, 0.1% Triton X-100, and 0.01% wt/vol gelatin. Thermal cycling consisted of 3 min denaturation at 94 C, followed by 30 cycles of 30 sec/94 C, 30 sec/55 C, 30 sec/72 C and a final incorporation at 72 C for 5 min. PCR products were analyzed by electrophoresis on a 6% polyacrylamide denaturing gel (Sequagel, National Diagnostics, Atlanta, GA) on an ALFexpress automated DNA sequencer from Amersham Pharmacia Biotech (Piscataway, NJ) (8).
Case report The patient was a male Caucasian born to nonconsanguineous, healthy parents; the family history is unremarkable. Mother was 31 yr old at the time of delivery. The boy was small for gestational age: birth weight was 1630 g at 38 weeks and 1 day. Fetal distress urged a caesarian
1164
ISODISOMY AND CONGENITAL HYPOTHYROIDISM
FIG. 1. A, DGGE pattern of the proband and his family. Starting from the left: lane 1, father; lane 2, mother; lane 3, sister; and lane 4, proband. In lane 2, the two bands represent mother’s heterozygosity and the last (fourth) lane represents the proband’s homozygosity for the mutation in the TPO gene. B, Genomic DNA sequence analysis. Assemblage of part of the TPO exon 14 DNA sequence. I:1, Father; I:2, mother; II:1, sister; and II:2, proband. WT sequences are I:1 and II:1; I:2 is heterozygous for the ⌬T2512 mutation; and II:2 is homozygous for ⌬T2512. section. However the neonate was vigorous after birth with Apgar scores of 8 and 9 after 1 and 5 min, respectively. Physical examination was unremarkable except for a preauricular skin tag on the left side. No preauricular skin tags were present in other family members. The renal ultrasound in the patient was unremarkable. Serious feeding problems in the first week of life prompted early assessment of thyroid function. Very low plasma free T4 and T4 levels, extremely elevated plasma TSH and elevated plasma Tg concentrations were found (Table 1). The ra-
1165
FIG. 2. Location of the genetic markers used for chromosome 2. Details of the STR markers from the Genome Database as of January 25, 2000 (http//:www.gdb.org). The hatched part of the bar to the right represents the length of the isodisomy of 2p confirmed by homozygosity of the markers; the unhatched part could also be involved in the isodisomy, but two markers (D2S2216 and D2S113) were not informative. dioiodide imaging study (1 MBq 123I⫺ iv, followed by measurements of the uptake in the thyroid every 30 min, and administration of 100 mg NaClO4 iv at 120 min) documented a normally located thyroid gland
1166
BAKKER ET AL.
JCE & M • 2001 Vol. 86 • No. 3
TABLE 1. Base line laboratory data in the propositus Venous blood: (day 7 pp)
Reference values
T4 15 nmol/L T3 0.75 nmol/L TSH 1840 mU/L FT4 1.6 pmol/L TBG 500 nmol/L Tg 2090 pmol/L
120 –220 nmol/L 1.5–3.0 nmol/L ⬍10 mU/l 12–25 pmol/L 380 –750 nmol/L 20 –375 pmol/L
with a high uptake (above 20%) and an immediate and complete release of the intrathyroidal radioiodine after NaClO4 administration. The expressed CH, elevated Tg concentrations, and complete release of 123I⫺ after NaClO4 administration were consistent with the diagnosis of TIOD. The now 5-yr-old boy has developed well and showed catch-up growth; at the time of this report, he is using thyroxine supplementation and his condition is excellent.
Results
The propositus was clinically diagnosed with TIOD, suggesting TPO dysfunction. The TPO gene (all exons and intron/exon boundaries) was screened for mutations by DGGE and in exon 14, a pattern consistent with a homozygous mutation was found (Fig. 1A); on sequence analysis, a homozygous deletion of one T at nucleotide position 2512 (codon 808) was identified. The mother appeared to be heterozygous for ⌬T2512, whereas in the TPO genes of the father and the sister this mutation was absent, consistent with the DGGE results (Fig. 1B). Additional DNA studies through typing with polymorphic short tandem repeat (STR) markers confirmed paternity, except for an aberrant haplotype on chromosome 2. Figure 3 demonstrates that 14 markers for 2p, from D2S319 at 2p24.3 to D2S2333 at 2p12, were homozygous and derived from one maternal chromosomal homolog. We concluded that maternal partial isodisomy is present with a breakpoint between 2p12 (D2S2333) and 2p11.2 (D2S274), represented by the bar to the right of Fig. 2. The hatched part of the bar represents the length of the isodisomy of 2p confrimed by the homozygosity of the STR markers. The unhatched part of the bar could be involved in the isodisomy, but the markers D2S2216 and D2S113 were not informative. Eleven STR markers used for chromosome arm 2q showed in the proband a (normal) distribution of maternal and paternal markers (Fig. 3). A total of 28 STR markers was used for chromosome 2. Discussion
The patient described presented with severe CH, caused by a TIOD, due to mutations in the TPO gene. TPO is a key enzyme in the synthesis of thyroid hormone and essential for iodide organification at the apical border of the thyroid follicular cells. In this patient, we identified a homozygous mutation, a ⌬T2512 in exon 14 of the patient’s TPO alleles. This frameshift mutation leads to a premature termination signal in exon 14. Previous in vitro analysis of a similar frameshift mutation in exon 14 (ins C 2505–2511) resulted in an inactive enzyme (7). The inheritance pattern of mutated TPO alleles in this family was not autosomal recessive as usual. Heterozygosity for the mutation was only detected in the mother. Because
FIG. 3. Pedigree showing the pattern of the genetic markers used for chromosome 2 in the proband and his relatives. Details of the STR markers in the proband, his parents, and sister. I:1, Father; I:2, mother; II:1, sister; and II:2, proband.
STR markers confirmed paternity, the proband either had a maternal isodisomy for (part of) chromosome 2 or an unbalanced karyotype due to a paternal chromosome anomaly involving chromosome 2p, leading to hemizygosity for the TPO locus in the proband. Both the patient and his parents had normal karyotypes, which excluded major structural chromosomal abnormali-
ISODISOMY AND CONGENITAL HYPOTHYROIDISM
ties. By means of deduction, we concluded that the patient had UPD for chromosome 2p. Dosage analysis on Southern blotting (9) confirmed the presence of two copies of the TPO gene (data not shown). UPD represents an imbalance in the distribution of paternal and maternal chromosomes in the offspring. It is defined as the presence (in a diploid individual) of two homologs of a specific chromosome pair inherited from only one parent (10). This condition can be complete or partial. Uniparental heterodisomy is the condition where both chromosome homologs in the offspring originate from one parent but are different from each other. If two identical copies of one single parental homolog are present, the condition is called uniparental isodisomy. The first case of UPD was described by Engel (11), who stated that the consequences on the phenotype may result from three potentially harmful effects: interference with genomic imprinting, (occasionally) vestigial aneuploidy (unbalanced karyotype) from which UPD initially may have originated, and isodisomy. In isodisomy, there is not only the risk for a disturbance due to imprinting, but the two pairs of homologs are identical creating homozygosity for a large region of a certain chromosome, with an associated increased risk for recessive disorders (11). The latter is the cause for the TIOD in the patient presented. UPD of chromosome 2 has been described five times thus far, all were maternal in origin, and two out of five cases involved isodisomy (12). Clinical features are listed in Table 2; it is of note that our patient has so few clinical features while patients of other authors are often more severely affected. Currently, different mechanisms are considered responsible for UPD, the most important ones being trisomy rescue, monosomy rescue, gametic complementation, and somatic TABLE 2. Clinico-pathologic features associated with (maternal) UPD of chromosome 2 Most frequently described: • (Severe to mild) intra uterine growth retardation (IUGR); • (Mostly severe) Oligohydramnios • Oligohydramnios sequence (e.g. pulmonary hypoplasia, nasal deviation, ankle deformation, Joint contractures) Less frequently described: Condition • Hypospadias (N⫽3) • Hypothyroidism (N⫽2) • Bronchopulmonary dysplasia (N⫽2) • Hyaline membrane disease (N⫽1) • Renal failure (N⫽1) • Vesico-ureteric reflux (N⫽1) • Patent ductus arteriosis (N⫽1) • Congenital pyloric stenosis (N⫽1) • Hiatus hernia (N⫽1) • Undescended testes (N⫽1) • Pectus carinatum (N⫽1)
Author(s): Johnston (22), Hansen (23), Bernard (24) Bakker (this study), Harrison (19) Harrison (19), Johnston (22) Harrison (19) Webb (25)
Bernard (24) Johnston 22
1167
(mitotic) recombination (13). According to several authors, UPD in humans is caused primarily by meiotic nondisjunction events (14, 15). However, in some cases somatic (mitotic) recombination events are also described as a cause for UPD (15–17). The majority of the cases appear to be associated with advanced maternal age, as is found in meiotic nondisjunction in general. In the presented case, maternal age was not advanced (31 yr). Our patient has a partial maternal isodisomy for 2p, whereas the 2q homologs are derived from both parents (see Fig. 3). Therefore, in this patient the recombination has to be postzygotic (mitotic). This could be due to recombination in a trisomic cell, followed by trisomy rescue or loss of paternal chromosome 2p followed by reduplication of maternal chromosome 2p. Postzygotic recombination in a diploid cell can result in mosaisicm with partial isodisomy (17). We were not able to test other tissues than periferal lymphocytes, but the presence of TIOD in the patient indicates that isodisomy of chromosome 2 is at least present in thyroid tissue. To the best of our knowledge, partial isodisomy for chromosome 2p as described in this report, has not been described before. This finding has important consequences for genetic counseling of these parents: usually parents of a child with a TIOD will be given a 25% recurrence risk, and in certain cases prenatal diagnostics through molecular studies will be offered. Because the recurrence risk for partial UPD is very low, the risk for TIOD in further offspring for this couple is negligible. With regard to this family, there is no need to perform prenatal studies during future pregnancies. The pathogenesis of the prenatal growth retardation and preauricular skin tag in the proband remains uncertain. Intrauterine growth retardation is usually not present in children born with CH including the ones with TIOD. We have analyzed a group of 45 newborns with TIOD, and their birth weight is not different from the rest of the population (17a). Explanations for the intrauterine growth retardation in this patient are homozygosity for other genes on chromosome 2p or an imprinting effect. An imprinting map of the human genome suggested that chromosome 2 might have imprinting effects (14, 18). This is based on the homology of human chromosome 2 with mouse chromosomes 2 and 12, which have shown to be imprinted. However, genes imprinted in mice are not necessarily imprinted in humans (12). One case report describing a newborn with UPD2mat, ascertained through trisomy mosaicism in amniotic fluid, also showing intra uterine growth retardation, hypothyroidism (etiology not mentioned) and hyaline membrane disease/bronchopulmonary problems (19), might be an indication of genomic imprinting for chromosome 2 (14). However, another patient with maternal isodisomy and two isochromosomes [i(2p) and i(2q)] was completely normal (20), which argues against maternally imprinted gene(s) on chromosome 2. According to McKusick’s “Mendelian Inheritance in Man” (http://www.ncbi.nlm.nih.gov/Omim), there is no condition on chromosome 2p associated with skin tags. In the cytogenetic literature, a duplication of 2p25–21 has been described in a patient with among others preauricular skin tags (21), indicating that genes on 2p can give rise to tag formation
1168
BAKKER ET AL.
once disturbed in their function. However, the patient described in the cytogenetic literature had many other pathological conditions, a big contrast to our patient with one minor dysmorphic feature. These patients with (partial) duplication or (partial) deletion of 2p show in general growth retardation, mental retardation, and many dysmorphic features. Some of these patients also have low-set, malformed ears, but no preauricular skin tags (Human Cytogenetics Database 1992). Regarding our patient homozygosity for other genes on 2p, or genomic imprinting of 2p as an explanation for the unilateral preauricular ear tag cannot be excluded. In conclusion: The patient described has severe congenital hypothyroidism caused by the dyshormonogenesis TIOD, which in turn is caused by a mutation in the TPO gene. Due to partial maternal isodisomy of the short arm of chromosome 2, the TPO gene mutation was reduced to homozygosity. In patients with these autosomal recessive thyroid disorders, it remains important to confirm heterozygosity for the causative mutation in both parents because this has a significant effect on genetic counseling. A role for genomic imprinting of chromosome 2p cannot be discarded by this study. However, these data show that partial maternal isodisomy of chromosome 2p (2pter–2p12) is compatible with a minimal influence on normal development. Acknowledgments Dr. Frank Baas and Dr. Marcel Mannens are gratefully acknowledged for reviewing the manuscript and their contribution to the discussion on the mechanism(s) involved in this form of UPD. Words of thanks also to Martin Thomas Young for editing the article regarding English and Jose Willemsen for secretarial assistance.
References 1. Vulsma T. Etiology and pathogenesis of congenital hypothyroidism. Evaluation and examination of patients detected by neonatal screening in the Netherlands. Academic Thesis 1990; University of Amsterdam. 2. De Vijlder JJM, Vulsma T. 1996 Hereditary metabolic disorders causing hypothyroidism. In: Braverman LE, Utiger RD, eds. Werner and Ingbar’s the thyroid: a fundamental and clinical text. 7th ed. Philadelphia: LippincottRaven; 749 –755. 3. Abramowicz MJ, Tarkovnik HM, Varela V, et al. 1992 Identification of a mutation in the coding sequence of the human thyroid peroxidase gene causing congenital goiter. J Clin Invest. 90:1200 –1204. 4. Bikker H, Den Hertog MT, Baas F, Gons MH, de Vijlder JJM. 1994 A 20-basepair duplication in the human thyroid peroxidase gene results in a total iodide organification defect and congenital hypothyroidism. J Clin Endocrinol Metab. 79:248 –252. 5. Bikker H, Vulsma T, Baas F, de Vijlder JJM. 1995 Identification of five novel inactivating mutations in the human thyroid peroxidase gene by denaturing gradient gel electrophoresis. Hum Mutat. 6:9 –16. 6. Taurog A. 1996 Hormone synthesis: thyroid iodine metabolism. In: Braverman
JCE & M • 2001 Vol. 86 • No. 3
LE, Utiger RD, eds. Werner and Ingbar’s the thyroid: a fundamental and clinical text. 7th ed. Philadelphia: Lippincott-Raven; 749 –755. 7. Bikker H, Baas F, De Vijlder JJM. 1997 Molecular analysis of mutated thyroid peroxidase detected in patients with total iodide organification defects. J Clin Endocrinol Metab. 82:649 – 653. 8. Benecke M. 1998 Random amplified polymorphic DNA (RAPD) typing of necrophageous insects (diptera, coleoptera) in criminal forensic studies: validation and use in practice. Forensic Sci Int. 98:157–168. 9. Hensels GW, Janssen EA, Hoogendijk JE, Valentijn LJ, Baas F, Bolhuis PA. 1993 Quantitative measurement of duplicated DNA as a diagnostic test for Charcot-Marie-Tooth disease type 1a. Clin Chem. 39:1845–1849. 10. Engel E. 1998 Uniparental disomies in unselected populations. Am J Hum Genet. 63:962–966. 11. Engel E. 1980 A new genetic concept: uniparental disomy and its potential effect, isodisomy. Am J Genet. 6:137–143. 12. Kotzot D. 1999 Abnormal phenotypes in uniparental disomy (UPD): fundamental aspects and a critical review with bibliography of UPD other than 15. Am J Med Genet. 82:265–274. 13. Berend SA, Feldman GL, McCaskill C, Czarnecki P, Van Dyke DL, Shaffer LG. 1999 Investigation of two cases of paternal disomy 13 suggests timing of isochromosome formation and mechanisms leading to uniparental disomy. Am J Med Genet. 82:275–281. 14. Ledbetter DH, Engel E. 1995 Uniparental disomy in humans: development of an imprinting map and its implications for prenatal diagnosis. Hum Mol Genet. 4:1757–1764. 15. Robinson WP, Kuchinka BD, Bernasconi F, et al. 1998 Maternal meiosis I non-disjunction of chromosome 15: dependence of the maternal age effect on level of recombination. Hum Mol Genet. 7:1011–1019. 16. Dutly F, Baumer A, Kayserili H, et al. 1998 Seven cases of WiedemannBeckwith syndrome, including the first reported case of mosaic paternal isodisomy along the whole chromosome 11. Am J Med Genet. 79:347–353. 17. Bischoff FZ, Feldman GL, McGaskill C, Subramanian S, Hughes MR, Schaffer LG. 1995 Single cell analysis demonstrating somatic mosaicism involving 11p in a patient with paternal isodisomy and Beckwith-Wiedemann syndrome. Hum Mol Genet. 4:395–399. 17a.Bakker B, Bikker H, Vulsma T, De Randamie JES, Wieclijk BM, de Vijlder JJM. 2001 Two decades of screening for congenital hypothyroidism in The Netherlands: TPO mutations in total iodide organification defects. J Clin Endocrinol Metab. In press. 18. Morison IM, Reeve AE. 1998 A catalogue of imprinted genes and parent-oforigin effects in humans and animals. Hum Mol Genet. 7:1599 –1609. 19. Harrison K, Eisenger K, Anyane-Yeboa K, Brown S. 1995 Maternal uniparental disomy of chromosome 2 in a baby with trisomy 2 mosaicism in amniotic fluid culture. Am J Med Genet. 58:147–151. 20. Bernasconi F, Karagu¨zel, Celep F, et al. 1996 Normal phenotype with maternal isodisomy in a female with two isochromosomes: i(2p) and i(2q). Am J Hum Genet. 59:1114 –1118. 21. Heathcote JG, Sholdice J, Walton JC. 1991 Anterior segment mesenchymal dysgenesis associated with partial duplication of the short arm of chromosome 2. Can J Ophthalmol. 26:35– 43. 22. Johnston KM, Baker JC, Egli CA, McCaskill C, Schaffer LG. 1996 Maternal uniparental isodisomy of chromosome 2 in a child with growth retardation, hypospadias, and a cytogenetic abnormality. 46,XY, i(2) (p10), i (2) (q10) Am J Hum Genet. 59(Suppl):A95 (Abstract 518). 23. Hansen WF, Bernard LE, Langlois S, et al. 1997 Maternal uniparental disomy of chromosome 2 and confined placental mosaicism for trisomy 2 in a fetus with intrauterine growth restriction, hypospadias, and oligohydramnios. Prenat Diagn. 17:443– 450. 24. Bernard LE, Kalousek DK, Langlois S, et al. 1995 Confined placental mosaicism for trisomy 2 with fetal maternal uniparental disomy of chromosome 2. Am J Hum Genet. 57:261 (Abstract). 25. Webb AL, Sturgiss S, Warwicker P, Robson SC, Goodship JA, Wolstenholme J. 1996 Maternal uniparental disomy for chromosome 2 in association with confined placental mosaicism for trisomy 2 and severe intrauterine growth retardation. Prenat Diagn. 16:958 –962.
