American Journal of Medical Genetics 43:791-795 (1992)
Pericentric Inversion of Chromosome 16 in a Large Kindred: Spectrum of Morbidity and Mortality in Offspring Diana W. Bianchi, Robert D. Nicholls, Kathryn A. Russell, Wayne A. Miller, Marvin Ellin, and Janice M. Lage Divisions of Genetics (D.W.B., R.D.N., M.E.) and Newborn Medicine (D.W.B.), The Children’s Hospital, Boston, Massachusetts, Department of Pathology (K.R., J.M.L.), Brigham and Women’s Hospital, Boston, Massachusetts, and Department of Obstetrics and Gynecology (W.A.M.1, Tufts University of School of Medicine, Boston, Massachusetts
Constitutional pericentric inversions of chromosome 16 are rare in the general population. We report here a large kindred who carry an inv(16)(p13q22)rearrangement. In general, individuals with the inv(l6) are in good health but prone to reproductive loss. Two different types of recombinant offspring were identified in this family and analyzed at the molecular level using probes from the or-globin and polycystic kidney disease loci. Both were associated with serious major malformations. 0 1992 Wiley-Liss, Inc.
KEY WORDS: chromosome 16 pericentric inversion, chromosome 16 duplication and deletion, reproductive loss INTRODUCTION Constitutional pericentric inversions of chromosome 16 are exceedingly rare despite recognized fragile sites at p13 and q22 [Kaiser, 19841. An acquired abnormality, inv(16)(p13q22),has been reported in a subset of patients with acute nonlymphocytic leukemia [LeBeau et al., 1983; Tantravahi et al., 1984; Holmes et al., 19851. The clonal rearrangements, seen in cells from bone marrow aspirates, are associated with a particular clinical presentation that includes eosinophilia, abnormal eosinophil morphology, a propensity for CNS relapse, and a favorable prognosis. An apparently identical pericentric inversion has been described in tumor cells (but not
Received for publication April 18, 1991; revision received September 25, 1991. Address reprint requests to Diana W. Bianchi, M.D., Division of Genetics, The Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. Present address of R.D. Nicholls: Department of Neuroscience and Division of Genetics, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610.
0 1992 Wiley-Liss, Inc.
peripheral blood leucocytes) in patients with colon cancer [Shabtai et al., 19871.This has prompted speculation that an oncogene may be activated as a result of the cytogenetic rearrangement. Here we report on a large family, consisting of many members who carry an inv(16)(p13q22)rearrangement. None of them has had leukemia or solid tumors. With the exception of 2 males who had neonatal jaundice and mild developmental delay, relatives with the inv(l6) are asymptomatic except for reproductive loss. Here we describe the spectrum of anomalies seen in 4 different offspring born to 3 sisters who carry the inv(l6).
CLINICAL REPORTS The pedigree is shown in Figure 1.The initial propositus was individual 111-6. A genetic consultation was obtained in 1984 because of neonatal hyperbilirubinemia and a family history of neonatal hepatitis. During his evaluation, a chromosome abnormality, inv(16) (p13q221, was identified. This prompted a more extensive study of his family. Patient 111-1 This male infant, born in 1972, was the 1.35 kg product of a 36-week uncomplicated pregnancy born to a then 23-year-oldG1 woman. He had growth retardation, generalized laxity of the skin, and abnormal appearance with bulging eyes, a sharp pointed nose, and absence of earlobes. His course was complicated by direct hyperbilirubinemia, hepatosplenomegaly, anemia, thrombocytopenia, and failure t o thrive. He died a t age 58 days of liver failure. Postmortem studies failed to elucidate a cause or pathogenesis for the liver disease. Chromosomes (Giemsa stained, not banded) were reported as normal. Patient 111-10 This male infant, born in 1982, was the 3 kgproduct of a 41-week gestation born to a then 30-year-old, G3P1, Sab 1, blood group B+ woman whose pregnancy was complicated by vaginal bleeding at 5 months, and asthma, treated with aminophylline. At the time of the pregnancy it was not known that the mother carried the
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I
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5 1
Fig. 1. Pedigree ofthe family. Key to symbols: 00,Chromosomes not tested; 0 (3inv 16 (p13q22);00, normal chromosomes; 0 ,rec 16.
inv(l6). The infant was delivered vaginally and had meconium stained amniotic fluid. Apgar scores were 6 at 1min and 7 at 5 min. He had severe cyanosis, a grade 216 systolicheart murmur, hypospadias, and a left club foot. Despite mechanical ventilation with 100% oxygen, the infant remained hypoxemic, and was transferred to an intensive care unit. Cardiac catheterization showed primary pulmonary hypertension, patent ductus arteriosus, an atrial septal defect, a pulmonary artery that was larger than the aorta, a thickened left ventricle, and tricuspid regurgitation. He remained persistently hypoxemic and died at age 15 days. Chromosome studies were not obtained. An autopsy was not performed. Patient 111-12 This female infant, born in 1985,was the sib of patient 111-10.She was the 2.8 kg product of a 41-week gestation born to a then 33-year-oldG5P2 Sab2 woman who again took aminophylline for asthma. During the third trimester of this pregnancy, the mother (11-5) was identified as having the inv(l6) as a result of the family study. Prenatal cytogenetic diagnosis was not offered due to the late stage of gestation. Delivery was normal, spontaneous vaginal with no evidence of fetal distress other than meconium stained amniotic fluid. The infant was cyanotic and required intubation and mechanical ventilation in the delivery room. Apgar scores were 3 at 1min and 7 at 5 min. Her physical exam was remarkable only for cyanosis and a grade 216 systolic heart murmur. He appearance suggested a Potter face (Fig. 2). An echocardiographic examination documented a large patent ductus arteriosus, right to left shunting through the foramen ovale, but no structural abnormalities. The infant remained profoundly hypoxemic and did not respond to therapy including chest tube placement for pneumothorax. She died at age 6 hr. The autopsy showed a severe preductal coarctation of the aorta, marked cardiomegaly with right ventricular hypertrophy, atrial
Fig. 2. Postmortem photograph of 111-12, showing the Potter-like face.
septal defect, secundum type, and pulmonary hypoplasia. There was myocyte hypertrophy in the heart. The thymus and adrenal glands evinced acute involutional changes. The lungs were atelectatic and showed focal congenital aspiration pneumonia with intraalveolar hemorrhages. The liver was normal by microscopy.
Fetus 111-17 This male fetus, delivered in 1988, was the product of a 23-week gestation to a 33-year-old G5P4 woman who
Pericentric Inversion of Chromosome 16 presented a t 21 weeks for gestational dating. At ultrasonographic examination, massive hydrocephalus was noted, and a peripheral umbilical blood chromosome analysis was performed. After the diagnosis of a chromosome abnormality was made (see laboratory studies), the mother informed her physician that she carried the inv(l6). She elected to terminate the pregnancy. The normally formed male fetus had a single umbilical artery, hydrocephalus of unknown pathogenesis, no structural abnormalities of the heart, and normal liver by microscopy. No gross malformations of the cerebellum or cerebral aqueduct were seen, but fragmentation of the brain stem and cerebellum precluded further anatomic evaluation.
LABORATORY INVESTIGATIONS Cytogenetics As indicated above, 111-1 and 111-10 died before the inv(l6) was identified in the family. However, in 111-12, the Giemsa banded karyotype was 46,XXrec(lG)dup (q22-q24)inv(l6)(pl3q22). The umbilical blood sample on 111-17produced an apparently identical recombinant chromosome: 46,XYrec(l6)dup(q22-24)inv(16)(p13q22) (Fig. 3). Molecular Analysis Cytogenetic analysis of this family identified breakpoints within bands 1 6 ~ 1 and 3 16q22 for the inverted or recombinant chromosomes. DNA probes that map close to or within the chromosome 16 bands that appeared to be rearranged were selected to permit molecular characterization of the recombinant chromosomes. The probes used were a-globin and polycystic kidney disease 1 (PKD1) loci, which map to 16~13.3[Higgs et al., 1989; Germino et al., 19901, and the metallothionein gene, a t 16q22 [Karin et al., 1984; Le Beau et al., 19851. DNA was obtained from all individuals in the family who had previously been karyotyped. DNA was prepared by conventional methods [Sambrook et al., 19891. As samples were available from 3 generations, it was possible to determine both phase and specific haplotypes for each individual in this family, thus permitting detection of meiotic crossovers in offspring. The metallothionein gene probes pMt-IIa (EcoRI) and pHSI (TaqI) [Karin et al., 19841 showed no abnormalities in band size or intensity for the bands corresponding to the cluster of genes mapped to 16q22. Probes specific for a-globin were initially selected for investigation of 16p. Psta detects the adult a1 and a2 genes on 23-kb EcoRI fragments in normal human DNA. With SstI digests, 2 alleles (a1 + 3’ HVR), comprising 14-16 kb, and 4-kb (a2) fragment, occur in normal individuals. The 3’ HVR is a variable number of tandem repeats (VNTR) locus that is highly polymorphic in human populations. Most individuals show heterozygosity 8 kb 3’ to the a1 gene [Higgs et al., 19891.A fragment of 20 kb (EcoRI) or a single 14-kb band (SstI) were obtained for 111-12 (data not shown), indicative of a 3.7-kb deletion fusing a2 and a1 into a single gene that represents the - 2 ’1 - - a-thalassemia genoiype [Higgs et al., 19891. The intensity and lack of a second
793
11-5
111-12
111-17
Fig. 3. Photograph of chromosome 16 pairs in 3 relatives. In each case, the normal chromosome 16 is on the left. Individual 11-51demonstrates the inv(l6) on the right. Individual 111-12 and fetus 111-17 demonstrate an apparently identical recombinant chromosome 16 on the right.
a-specific fragment spanning the 3’ HVR suggest that the entire a-globin locus is deleted from the chromosome 16 homologue. To further study the molecular extent of deletion for chromosome 1 6 ~ 1in 3 111-12and the molecular nature of the rearrangement in 111-17, we analyzed the segregation of three VNTR loci in this family: the 3’ HVR; the 5’ HVR, located about 100 kb telomeric to the adult a-globin genes [Wilkie et al., 19901 (Fig. 4); and EKMDA2 from the PKDl locus [Germino et al., 19901 (Table I). PKDl lies approximately 1Mb centromeric to a-globin [Harris et al., 19901. All 3 loci are deleted from 111-12. In contrast, 111-17 is deleted for the 2 a-globin probes but heterozygous for EKMDA2, and hence intact for the PKDl locus. The individual molecular haplotypes shown in Table I were determined by assuming the minimum number of meiotic crossovers. The grandfather (1-2)was deceased, so his haplotypes were reconstructed. Individuals 11-2, 11-7,and their offspring with inv(l6) placed the U allele of the 5’ HVR locus and the 3.9-kb allele of the 3’ HVR locus on the inv(l6) chromosome of 1-2, since neither allele was present in the grandmother (1-11, who has normal chromosomes (Fig. 1).Conversely,the L allele of the 5’ HVR locus and the 3.7-kb allele of the 3’ HVR locus were inherited from 1-1.This is consistent with the results found in 11-3, who has normal chromosomes. Individual 11-3 further demonstrates that the normal chromosome 16 inherited from 1-2 contains the L 5’ HVR and 4.8-kb 3’ HVR alleles. All second generation offspring and 1-1have 1.9- and 1.7-kb alleles for EKMDA2. Individuals 111-4, 111-6, 111-13,111-14,and 111-15show that the 1.9-kb allele segregates with the inv(l6). The grandpaternal (1-2)haplotypes are L-4.8-1.9 (normal) and U-3.9-1.9 [inv(l6)1. The grandmaternal (1-1)haplotypes are L-3.7-1.7 and L-3.4-1.9,where the order ofloci is telomere-5’ HVR-3’ HVR-PKD1-centromere [Wilkie et al., 19911. Anormal meiotic crossover was identified in this family study. Individual 11-5 has the inv(l6) chromosome but not the inv(l6)5‘HVR allele (U) or the 3’ HVR allele (3.9 kb). The inv(l6) is, in this case, associated with the L-4.8-1.9 grandpaternal haplotype.
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kb
-4.8 -U L
-3.7 - 3.3
A Fig. 4. Molecular identification of chromosome 16p deletions in 111-12and 111-17, the two recombinant individuals. (A)Hybridization of TaqI digested DNA from III-12,111-8, and 11-5with the 3’HVRprobe. 111-8 inherits the paternal 3.3-kb allele and the maternal 3.7-kb allele. 111-12 only inherits the paternal 3.3-kb allele, but no maternal allele, indicating a deletion of maternal origin. (B) Hybridization of TaqI digested DNA from family members with the 5’ HVR. 111-17 has a maternal allele only, indicating a deletion of paternal or postzygotic origin (see text).
DISCUSSION
TABLE I. Molecular Haplotypes for 16p13* -
I- 1 1-2 (implied) 11-2 11-3 11-5 11-5 (spouse)
5’ HVR L L U* L U* L L L3.7 L* L U
11-7 (spouse) 111-4 111-6 111-8
U* L U U U* L U* L U L U U* U U* U U* U
3.3 3.9* 5.2 3.9* 5.2 3.9* 5.2
L
3.7
L
3.7
-
111-12 111-13 111-14 111-15
-
111-17 or
4.8*
3.7 3.3 4.8 3.9* 3.7 5.2 3.9 3.9* 3.7 3.9* 3.95 3.3 3.7
2
11-7
HVR 3.7 3.4 3.9* 4.8 3.9* 3.7 4.8 3.7
3’ -~
-
-
-
-
*Alleles marked by an asterisk indicate the (inv)l6
EKMDA2
~-
1.7 1.9 1.9” 1.9 1.9* 1.7 1.9 1.7 1.9* 1.7 1.9 1.9 1.9* 1.7 2.5 2.4 1.9* 1.9 1.9* 1.9 1.9 1.7 -
1.9 1.9* 2.5 1.9* 2.5 1.9* 2.5 2.4 1.9 1.9 2.4
We have presented a clinical description of 3 infants who died perinatally and one abortus who were delivered to mothers with inv(16)(p13q22).One ofthe infants (111-10) was not karyotyped and one (111-1)had a “normal” karyotype in the prebanding era. The other two (111-12 and 111-17)had apparently identical recombinant chromosome 16s by cytogenetic analysis but they differed with the use of molecular techniques. It is tempting to speculate that the sibs 111-10 and 111-12 had very similar recombinant chromosomes since both were so alike in phenotype and clinical course. We are aware of only 2 other children with similar recombinant chromosome 16s. The first is a n ll-yearold boy with dup(p13.1 p13.3)del (q22 q24) [Ionasescu, 19871. He has profound growth and mental retardation, microcephaly, agenesis of the corpus callosum, loss of brain parenchyma, grand ma1 seizures, sensorineural hearing loss, blepharophimosis, cataracts, thoracolumbar kyphoscoliosis, and a myopathy. The second is a n 11month-old male with rec(l6)dupqinvl6 (qter >p13.3: : q22.1 >qter) [Warburton and Anyane-Yeboa, 19901. He has failure to thrive, normal head growth, left ptosis, tracheomalacia with gastroesophageal reflux, abnormal swallowing coordination, cleft tongue, right inguinal hernia, and bilateral club feet. He has undergone catheterization for evaluation of persistent cyanosis, showing severe pulmonary artery hypertension and a right to left shunt through a n atrial septa1 defect. In both cases, the mother carries a n inv(16)(p13q22). The families are not related to each other. Molecular analysis, in one case (111-121,confirmed the cytogenetic results. In the other recombinant (111-17), molecular analysis appears to imply a smaller deletion of 16p. The inv(l6) (p13q22) characterized in this family predisposes to recombination during meiosis. One event (1-2 to 11-5) is a normal meiotic crossover, centromeric to
Pericentric Inversion of Chromosome 16 a-globin, that exchanges markers near the telomere of 16p on the inv(l6) chromosome. All other recombinations detected in this family are unequal, leading to an unbalanced karyotype and malformations in the fetus. The 16p breakpoint in 111-12 is centromeric to PKDl, whereas the 16p breakpoint in 111-17 has been mapped to the interval between a-globin and PKD1. Interestingly, a complicated genesis of the recombinant chromosome 16 in 111-17 is likely, since the pattern of maternal and paternal alleles for 16p markers suggests a meiotic exchange of distal loci on the inv(l6) during maternal meiosis, and a deletion of paternal a-globin loci. It is not known whether the apparent recombination on the paternal chromosome 16 is independent (meiotic)or influenced by the inv(l6) and thus a postzygotic (mitotic) event. Furthermore, it is of interest that 111-12 had a - a3.’/- - genotype for a globin. The - a3.7 chromosome is the same one as that commonly found in a-thalassemia [Higgs et al., 19891, whereas the - complete a-globin gene deletion (including the 3’ HVR sequence) has only been found in individuals with the a-thalassemidmental retardation syndrome [Weathera11 et al., 1981; Wilkie et al., 19901. Absence of three a-globin genes generally results in hemoglobin H disease. The presence of abnormal hemoglobin levels may have contributed to the hypoxemia seen in individuals 111-10 and 111-12. In the family described here, there are at least 6 young children in generation I11 who carry the inv(l6). They are all at risk for developing abnormal recombinants in their gametes. Although none of the individuals has yet reached reproductive age, we have met with all of their parents to provide genetic counseling. As tragically demonstrated in this family, prenatal diagnosis should be offered to any individual known to carry inv(l6).
ACKNOWLEDGMENTS The authors would like to thank the Cytogenetics laboratories at Brigham and Women’s Hospital and f i e natal Diagnostic Center (Lexington, MA) for karyotypes; Dr. Dale Reiser a t St. Margaret’s Hospital (Boston) and Dr. Harland Winter at Children’s Hospital (Boston) for patient referral; and Drs. D.R. Higgs, S.T. Reeders, M. Karin, and R. Richards for the chromosome 16 probes used in this study.
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REFERENCES Germino GG, Barton NJ, Lamb J, Higgs DR, Harris P, Xiao GH, Scherer G, NakamuraY, Reeders ST (1990): Identification ofa locus which shows no genetic recombination with the autosomal dominant polycystic kidney disease gene on chromosome 16. Am J Hum Genet 46:925-933. Harris PC, Barton NJ, Higgs DR, Reeders ST, Wilkie AOM (1990): A long-range restriction map between the a-globin complex and a marker closely linked to the polycystic kidney disease 1 (PKD1) locus. Genomics 7:195-206. Higgs DR, Vickers MA, Wilkie AOM, E’retorius I-M, Jarman AP, Weatherall DJ (1989): A review of the molecular genetics of the human a-globin gene cluster. Blood 73:1081-1104. Holmes R, Keating MJ, Cork A, Broach Y, ‘hujillo J, Dalton WT Jr, McCredie KB, Freireich E J (1985): A unique pattern of central nervous system leukemia in acute myelomonocytic leukemia associated with inv(16)(p13q22). Blood 651071-1078. Ionasescu V, Patil S, Hart M, Rhead W, Smith W (1987): Multiple congenital anomalies syndrome with myopathy in chromosome 16 abnormality. Am J Med Genet 26:189-194. Kaiser P (1984): Pericentric inversions: Problems and significance for clinical genetics. Hum Genet 68:l-47. Karin M, Eddy RL, Henry WM, Haley LL, Byers MG, Shows TB (1984): Human metallothionein genes are clustered on chromosome 16. Proc Natl Acad Sci USA 81:5495-5498. LeBeau MM, Larson RA, Bitter MA, Vardiman JW, Golomb HW, Rowley J D (1983): Association of a n inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia. N Engl J Med 309:603-636. LeBeau MM, Diaz MO, Karin M, Rowley J D (1985): Metallothionein gene cluster is split by chromosome 16 rearrangements in myelomonocytic leukemia. Nature (London) 313:709-711. Sambrook J, Fritsch EF, ManiatisT (1989): Molecular cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, pp 9.14-9.24. Shabtai F, Sternberg A, Klar D, Reiss R, Halbrecht I(1987):Inversion (16)(p13q22)in tumor cells of sigmoid colon. Cancer Genet Cytogenet 27:171-175. Tantravahi R, Schwenn M, Henkle C, Nell M, Leavitt PR, Griffin JD, Weinstein H J (1984): A pericentric inversion of chromosome 16 is associated with dysplastic marrow eosinophils in acute myelomonocytic leukemia. Blood 63:800-802. Warburton D, Anyane-Yeboa K (1990): Personal communication. Weatherall DJ, Higgs DR, Bunch C, Old JM, Hunt DM, Pressley L, Clegg JB, Bethlenfalvay NC, Sjolin S, Koler RD, Magenis E, Francis JL, Bebbington D (1981): Hemoglobin H disease and mental retardation: A new syndrome or a remarkable coincidence? N Engl J Med 305.607-612. Wilkie AOM, Buckle VJ, Harris PC, Lamb J , Barton NJ, Reeders ST, Lindenbaum RH, Nicholls RD, Barrow M, Bethlenfalvay NC, Hutz MH, Tolmie JL, Weatherall DJ, Higgs DR (1990):Clinical features and molecular analysis of the a thalassemiaimental retardation syndromes. I. Cases due to deletions involving chromosomes band 161313.3. Am J Hum Genet 46:1112-1126. Wilkie AOM, Higgs DR, Rack KA, Buckle VJ, Spurr NK, FischelGhodsian N, Ceccherini I, Brown WRA, Harris PC (19911: Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16. Cell 64595-600.
Pericentric Inversion of Chromosome 16 in a Large Kindred: Spectrum of Morbidity and Mortality in Offspring Diana W. Bianchi, Robert D. Nicholls, Kathryn A. Russell, Wayne A. Miller, Marvin Ellin, and Janice M. Lage Divisions of Genetics (D.W.B., R.D.N., M.E.) and Newborn Medicine (D.W.B.), The Children’s Hospital, Boston, Massachusetts, Department of Pathology (K.R., J.M.L.), Brigham and Women’s Hospital, Boston, Massachusetts, and Department of Obstetrics and Gynecology (W.A.M.1, Tufts University of School of Medicine, Boston, Massachusetts
Constitutional pericentric inversions of chromosome 16 are rare in the general population. We report here a large kindred who carry an inv(16)(p13q22)rearrangement. In general, individuals with the inv(l6) are in good health but prone to reproductive loss. Two different types of recombinant offspring were identified in this family and analyzed at the molecular level using probes from the or-globin and polycystic kidney disease loci. Both were associated with serious major malformations. 0 1992 Wiley-Liss, Inc.
KEY WORDS: chromosome 16 pericentric inversion, chromosome 16 duplication and deletion, reproductive loss INTRODUCTION Constitutional pericentric inversions of chromosome 16 are exceedingly rare despite recognized fragile sites at p13 and q22 [Kaiser, 19841. An acquired abnormality, inv(16)(p13q22),has been reported in a subset of patients with acute nonlymphocytic leukemia [LeBeau et al., 1983; Tantravahi et al., 1984; Holmes et al., 19851. The clonal rearrangements, seen in cells from bone marrow aspirates, are associated with a particular clinical presentation that includes eosinophilia, abnormal eosinophil morphology, a propensity for CNS relapse, and a favorable prognosis. An apparently identical pericentric inversion has been described in tumor cells (but not
Received for publication April 18, 1991; revision received September 25, 1991. Address reprint requests to Diana W. Bianchi, M.D., Division of Genetics, The Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. Present address of R.D. Nicholls: Department of Neuroscience and Division of Genetics, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610.
0 1992 Wiley-Liss, Inc.
peripheral blood leucocytes) in patients with colon cancer [Shabtai et al., 19871.This has prompted speculation that an oncogene may be activated as a result of the cytogenetic rearrangement. Here we report on a large family, consisting of many members who carry an inv(16)(p13q22)rearrangement. None of them has had leukemia or solid tumors. With the exception of 2 males who had neonatal jaundice and mild developmental delay, relatives with the inv(l6) are asymptomatic except for reproductive loss. Here we describe the spectrum of anomalies seen in 4 different offspring born to 3 sisters who carry the inv(l6).
CLINICAL REPORTS The pedigree is shown in Figure 1.The initial propositus was individual 111-6. A genetic consultation was obtained in 1984 because of neonatal hyperbilirubinemia and a family history of neonatal hepatitis. During his evaluation, a chromosome abnormality, inv(16) (p13q221, was identified. This prompted a more extensive study of his family. Patient 111-1 This male infant, born in 1972, was the 1.35 kg product of a 36-week uncomplicated pregnancy born to a then 23-year-oldG1 woman. He had growth retardation, generalized laxity of the skin, and abnormal appearance with bulging eyes, a sharp pointed nose, and absence of earlobes. His course was complicated by direct hyperbilirubinemia, hepatosplenomegaly, anemia, thrombocytopenia, and failure t o thrive. He died a t age 58 days of liver failure. Postmortem studies failed to elucidate a cause or pathogenesis for the liver disease. Chromosomes (Giemsa stained, not banded) were reported as normal. Patient 111-10 This male infant, born in 1982, was the 3 kgproduct of a 41-week gestation born to a then 30-year-old, G3P1, Sab 1, blood group B+ woman whose pregnancy was complicated by vaginal bleeding at 5 months, and asthma, treated with aminophylline. At the time of the pregnancy it was not known that the mother carried the
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Fig. 1. Pedigree ofthe family. Key to symbols: 00,Chromosomes not tested; 0 (3inv 16 (p13q22);00, normal chromosomes; 0 ,rec 16.
inv(l6). The infant was delivered vaginally and had meconium stained amniotic fluid. Apgar scores were 6 at 1min and 7 at 5 min. He had severe cyanosis, a grade 216 systolicheart murmur, hypospadias, and a left club foot. Despite mechanical ventilation with 100% oxygen, the infant remained hypoxemic, and was transferred to an intensive care unit. Cardiac catheterization showed primary pulmonary hypertension, patent ductus arteriosus, an atrial septal defect, a pulmonary artery that was larger than the aorta, a thickened left ventricle, and tricuspid regurgitation. He remained persistently hypoxemic and died at age 15 days. Chromosome studies were not obtained. An autopsy was not performed. Patient 111-12 This female infant, born in 1985,was the sib of patient 111-10.She was the 2.8 kg product of a 41-week gestation born to a then 33-year-oldG5P2 Sab2 woman who again took aminophylline for asthma. During the third trimester of this pregnancy, the mother (11-5) was identified as having the inv(l6) as a result of the family study. Prenatal cytogenetic diagnosis was not offered due to the late stage of gestation. Delivery was normal, spontaneous vaginal with no evidence of fetal distress other than meconium stained amniotic fluid. The infant was cyanotic and required intubation and mechanical ventilation in the delivery room. Apgar scores were 3 at 1min and 7 at 5 min. Her physical exam was remarkable only for cyanosis and a grade 216 systolic heart murmur. He appearance suggested a Potter face (Fig. 2). An echocardiographic examination documented a large patent ductus arteriosus, right to left shunting through the foramen ovale, but no structural abnormalities. The infant remained profoundly hypoxemic and did not respond to therapy including chest tube placement for pneumothorax. She died at age 6 hr. The autopsy showed a severe preductal coarctation of the aorta, marked cardiomegaly with right ventricular hypertrophy, atrial
Fig. 2. Postmortem photograph of 111-12, showing the Potter-like face.
septal defect, secundum type, and pulmonary hypoplasia. There was myocyte hypertrophy in the heart. The thymus and adrenal glands evinced acute involutional changes. The lungs were atelectatic and showed focal congenital aspiration pneumonia with intraalveolar hemorrhages. The liver was normal by microscopy.
Fetus 111-17 This male fetus, delivered in 1988, was the product of a 23-week gestation to a 33-year-old G5P4 woman who
Pericentric Inversion of Chromosome 16 presented a t 21 weeks for gestational dating. At ultrasonographic examination, massive hydrocephalus was noted, and a peripheral umbilical blood chromosome analysis was performed. After the diagnosis of a chromosome abnormality was made (see laboratory studies), the mother informed her physician that she carried the inv(l6). She elected to terminate the pregnancy. The normally formed male fetus had a single umbilical artery, hydrocephalus of unknown pathogenesis, no structural abnormalities of the heart, and normal liver by microscopy. No gross malformations of the cerebellum or cerebral aqueduct were seen, but fragmentation of the brain stem and cerebellum precluded further anatomic evaluation.
LABORATORY INVESTIGATIONS Cytogenetics As indicated above, 111-1 and 111-10 died before the inv(l6) was identified in the family. However, in 111-12, the Giemsa banded karyotype was 46,XXrec(lG)dup (q22-q24)inv(l6)(pl3q22). The umbilical blood sample on 111-17produced an apparently identical recombinant chromosome: 46,XYrec(l6)dup(q22-24)inv(16)(p13q22) (Fig. 3). Molecular Analysis Cytogenetic analysis of this family identified breakpoints within bands 1 6 ~ 1 and 3 16q22 for the inverted or recombinant chromosomes. DNA probes that map close to or within the chromosome 16 bands that appeared to be rearranged were selected to permit molecular characterization of the recombinant chromosomes. The probes used were a-globin and polycystic kidney disease 1 (PKD1) loci, which map to 16~13.3[Higgs et al., 1989; Germino et al., 19901, and the metallothionein gene, a t 16q22 [Karin et al., 1984; Le Beau et al., 19851. DNA was obtained from all individuals in the family who had previously been karyotyped. DNA was prepared by conventional methods [Sambrook et al., 19891. As samples were available from 3 generations, it was possible to determine both phase and specific haplotypes for each individual in this family, thus permitting detection of meiotic crossovers in offspring. The metallothionein gene probes pMt-IIa (EcoRI) and pHSI (TaqI) [Karin et al., 19841 showed no abnormalities in band size or intensity for the bands corresponding to the cluster of genes mapped to 16q22. Probes specific for a-globin were initially selected for investigation of 16p. Psta detects the adult a1 and a2 genes on 23-kb EcoRI fragments in normal human DNA. With SstI digests, 2 alleles (a1 + 3’ HVR), comprising 14-16 kb, and 4-kb (a2) fragment, occur in normal individuals. The 3’ HVR is a variable number of tandem repeats (VNTR) locus that is highly polymorphic in human populations. Most individuals show heterozygosity 8 kb 3’ to the a1 gene [Higgs et al., 19891.A fragment of 20 kb (EcoRI) or a single 14-kb band (SstI) were obtained for 111-12 (data not shown), indicative of a 3.7-kb deletion fusing a2 and a1 into a single gene that represents the - 2 ’1 - - a-thalassemia genoiype [Higgs et al., 19891. The intensity and lack of a second
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11-5
111-12
111-17
Fig. 3. Photograph of chromosome 16 pairs in 3 relatives. In each case, the normal chromosome 16 is on the left. Individual 11-51demonstrates the inv(l6) on the right. Individual 111-12 and fetus 111-17 demonstrate an apparently identical recombinant chromosome 16 on the right.
a-specific fragment spanning the 3’ HVR suggest that the entire a-globin locus is deleted from the chromosome 16 homologue. To further study the molecular extent of deletion for chromosome 1 6 ~ 1in 3 111-12and the molecular nature of the rearrangement in 111-17, we analyzed the segregation of three VNTR loci in this family: the 3’ HVR; the 5’ HVR, located about 100 kb telomeric to the adult a-globin genes [Wilkie et al., 19901 (Fig. 4); and EKMDA2 from the PKDl locus [Germino et al., 19901 (Table I). PKDl lies approximately 1Mb centromeric to a-globin [Harris et al., 19901. All 3 loci are deleted from 111-12. In contrast, 111-17 is deleted for the 2 a-globin probes but heterozygous for EKMDA2, and hence intact for the PKDl locus. The individual molecular haplotypes shown in Table I were determined by assuming the minimum number of meiotic crossovers. The grandfather (1-2)was deceased, so his haplotypes were reconstructed. Individuals 11-2, 11-7,and their offspring with inv(l6) placed the U allele of the 5’ HVR locus and the 3.9-kb allele of the 3’ HVR locus on the inv(l6) chromosome of 1-2, since neither allele was present in the grandmother (1-11, who has normal chromosomes (Fig. 1).Conversely,the L allele of the 5’ HVR locus and the 3.7-kb allele of the 3’ HVR locus were inherited from 1-1.This is consistent with the results found in 11-3, who has normal chromosomes. Individual 11-3 further demonstrates that the normal chromosome 16 inherited from 1-2 contains the L 5’ HVR and 4.8-kb 3’ HVR alleles. All second generation offspring and 1-1have 1.9- and 1.7-kb alleles for EKMDA2. Individuals 111-4, 111-6, 111-13,111-14,and 111-15show that the 1.9-kb allele segregates with the inv(l6). The grandpaternal (1-2)haplotypes are L-4.8-1.9 (normal) and U-3.9-1.9 [inv(l6)1. The grandmaternal (1-1)haplotypes are L-3.7-1.7 and L-3.4-1.9,where the order ofloci is telomere-5’ HVR-3’ HVR-PKD1-centromere [Wilkie et al., 19911. Anormal meiotic crossover was identified in this family study. Individual 11-5 has the inv(l6) chromosome but not the inv(l6)5‘HVR allele (U) or the 3’ HVR allele (3.9 kb). The inv(l6) is, in this case, associated with the L-4.8-1.9 grandpaternal haplotype.
794
Bianchi et al.
kb
-4.8 -U L
-3.7 - 3.3
A Fig. 4. Molecular identification of chromosome 16p deletions in 111-12and 111-17, the two recombinant individuals. (A)Hybridization of TaqI digested DNA from III-12,111-8, and 11-5with the 3’HVRprobe. 111-8 inherits the paternal 3.3-kb allele and the maternal 3.7-kb allele. 111-12 only inherits the paternal 3.3-kb allele, but no maternal allele, indicating a deletion of maternal origin. (B) Hybridization of TaqI digested DNA from family members with the 5’ HVR. 111-17 has a maternal allele only, indicating a deletion of paternal or postzygotic origin (see text).
DISCUSSION
TABLE I. Molecular Haplotypes for 16p13* -
I- 1 1-2 (implied) 11-2 11-3 11-5 11-5 (spouse)
5’ HVR L L U* L U* L L L3.7 L* L U
11-7 (spouse) 111-4 111-6 111-8
U* L U U U* L U* L U L U U* U U* U U* U
3.3 3.9* 5.2 3.9* 5.2 3.9* 5.2
L
3.7
L
3.7
-
111-12 111-13 111-14 111-15
-
111-17 or
4.8*
3.7 3.3 4.8 3.9* 3.7 5.2 3.9 3.9* 3.7 3.9* 3.95 3.3 3.7
2
11-7
HVR 3.7 3.4 3.9* 4.8 3.9* 3.7 4.8 3.7
3’ -~
-
-
-
-
*Alleles marked by an asterisk indicate the (inv)l6
EKMDA2
~-
1.7 1.9 1.9” 1.9 1.9* 1.7 1.9 1.7 1.9* 1.7 1.9 1.9 1.9* 1.7 2.5 2.4 1.9* 1.9 1.9* 1.9 1.9 1.7 -
1.9 1.9* 2.5 1.9* 2.5 1.9* 2.5 2.4 1.9 1.9 2.4
We have presented a clinical description of 3 infants who died perinatally and one abortus who were delivered to mothers with inv(16)(p13q22).One ofthe infants (111-10) was not karyotyped and one (111-1)had a “normal” karyotype in the prebanding era. The other two (111-12 and 111-17)had apparently identical recombinant chromosome 16s by cytogenetic analysis but they differed with the use of molecular techniques. It is tempting to speculate that the sibs 111-10 and 111-12 had very similar recombinant chromosomes since both were so alike in phenotype and clinical course. We are aware of only 2 other children with similar recombinant chromosome 16s. The first is a n ll-yearold boy with dup(p13.1 p13.3)del (q22 q24) [Ionasescu, 19871. He has profound growth and mental retardation, microcephaly, agenesis of the corpus callosum, loss of brain parenchyma, grand ma1 seizures, sensorineural hearing loss, blepharophimosis, cataracts, thoracolumbar kyphoscoliosis, and a myopathy. The second is a n 11month-old male with rec(l6)dupqinvl6 (qter >p13.3: : q22.1 >qter) [Warburton and Anyane-Yeboa, 19901. He has failure to thrive, normal head growth, left ptosis, tracheomalacia with gastroesophageal reflux, abnormal swallowing coordination, cleft tongue, right inguinal hernia, and bilateral club feet. He has undergone catheterization for evaluation of persistent cyanosis, showing severe pulmonary artery hypertension and a right to left shunt through a n atrial septa1 defect. In both cases, the mother carries a n inv(16)(p13q22). The families are not related to each other. Molecular analysis, in one case (111-121,confirmed the cytogenetic results. In the other recombinant (111-17), molecular analysis appears to imply a smaller deletion of 16p. The inv(l6) (p13q22) characterized in this family predisposes to recombination during meiosis. One event (1-2 to 11-5) is a normal meiotic crossover, centromeric to
Pericentric Inversion of Chromosome 16 a-globin, that exchanges markers near the telomere of 16p on the inv(l6) chromosome. All other recombinations detected in this family are unequal, leading to an unbalanced karyotype and malformations in the fetus. The 16p breakpoint in 111-12 is centromeric to PKDl, whereas the 16p breakpoint in 111-17 has been mapped to the interval between a-globin and PKD1. Interestingly, a complicated genesis of the recombinant chromosome 16 in 111-17 is likely, since the pattern of maternal and paternal alleles for 16p markers suggests a meiotic exchange of distal loci on the inv(l6) during maternal meiosis, and a deletion of paternal a-globin loci. It is not known whether the apparent recombination on the paternal chromosome 16 is independent (meiotic)or influenced by the inv(l6) and thus a postzygotic (mitotic) event. Furthermore, it is of interest that 111-12 had a - a3.’/- - genotype for a globin. The - a3.7 chromosome is the same one as that commonly found in a-thalassemia [Higgs et al., 19891, whereas the - complete a-globin gene deletion (including the 3’ HVR sequence) has only been found in individuals with the a-thalassemidmental retardation syndrome [Weathera11 et al., 1981; Wilkie et al., 19901. Absence of three a-globin genes generally results in hemoglobin H disease. The presence of abnormal hemoglobin levels may have contributed to the hypoxemia seen in individuals 111-10 and 111-12. In the family described here, there are at least 6 young children in generation I11 who carry the inv(l6). They are all at risk for developing abnormal recombinants in their gametes. Although none of the individuals has yet reached reproductive age, we have met with all of their parents to provide genetic counseling. As tragically demonstrated in this family, prenatal diagnosis should be offered to any individual known to carry inv(l6).
ACKNOWLEDGMENTS The authors would like to thank the Cytogenetics laboratories at Brigham and Women’s Hospital and f i e natal Diagnostic Center (Lexington, MA) for karyotypes; Dr. Dale Reiser a t St. Margaret’s Hospital (Boston) and Dr. Harland Winter at Children’s Hospital (Boston) for patient referral; and Drs. D.R. Higgs, S.T. Reeders, M. Karin, and R. Richards for the chromosome 16 probes used in this study.
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REFERENCES Germino GG, Barton NJ, Lamb J, Higgs DR, Harris P, Xiao GH, Scherer G, NakamuraY, Reeders ST (1990): Identification ofa locus which shows no genetic recombination with the autosomal dominant polycystic kidney disease gene on chromosome 16. Am J Hum Genet 46:925-933. Harris PC, Barton NJ, Higgs DR, Reeders ST, Wilkie AOM (1990): A long-range restriction map between the a-globin complex and a marker closely linked to the polycystic kidney disease 1 (PKD1) locus. Genomics 7:195-206. Higgs DR, Vickers MA, Wilkie AOM, E’retorius I-M, Jarman AP, Weatherall DJ (1989): A review of the molecular genetics of the human a-globin gene cluster. Blood 73:1081-1104. Holmes R, Keating MJ, Cork A, Broach Y, ‘hujillo J, Dalton WT Jr, McCredie KB, Freireich E J (1985): A unique pattern of central nervous system leukemia in acute myelomonocytic leukemia associated with inv(16)(p13q22). Blood 651071-1078. Ionasescu V, Patil S, Hart M, Rhead W, Smith W (1987): Multiple congenital anomalies syndrome with myopathy in chromosome 16 abnormality. Am J Med Genet 26:189-194. Kaiser P (1984): Pericentric inversions: Problems and significance for clinical genetics. Hum Genet 68:l-47. Karin M, Eddy RL, Henry WM, Haley LL, Byers MG, Shows TB (1984): Human metallothionein genes are clustered on chromosome 16. Proc Natl Acad Sci USA 81:5495-5498. LeBeau MM, Larson RA, Bitter MA, Vardiman JW, Golomb HW, Rowley J D (1983): Association of a n inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia. N Engl J Med 309:603-636. LeBeau MM, Diaz MO, Karin M, Rowley J D (1985): Metallothionein gene cluster is split by chromosome 16 rearrangements in myelomonocytic leukemia. Nature (London) 313:709-711. Sambrook J, Fritsch EF, ManiatisT (1989): Molecular cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, pp 9.14-9.24. Shabtai F, Sternberg A, Klar D, Reiss R, Halbrecht I(1987):Inversion (16)(p13q22)in tumor cells of sigmoid colon. Cancer Genet Cytogenet 27:171-175. Tantravahi R, Schwenn M, Henkle C, Nell M, Leavitt PR, Griffin JD, Weinstein H J (1984): A pericentric inversion of chromosome 16 is associated with dysplastic marrow eosinophils in acute myelomonocytic leukemia. Blood 63:800-802. Warburton D, Anyane-Yeboa K (1990): Personal communication. Weatherall DJ, Higgs DR, Bunch C, Old JM, Hunt DM, Pressley L, Clegg JB, Bethlenfalvay NC, Sjolin S, Koler RD, Magenis E, Francis JL, Bebbington D (1981): Hemoglobin H disease and mental retardation: A new syndrome or a remarkable coincidence? N Engl J Med 305.607-612. Wilkie AOM, Buckle VJ, Harris PC, Lamb J , Barton NJ, Reeders ST, Lindenbaum RH, Nicholls RD, Barrow M, Bethlenfalvay NC, Hutz MH, Tolmie JL, Weatherall DJ, Higgs DR (1990):Clinical features and molecular analysis of the a thalassemiaimental retardation syndromes. I. Cases due to deletions involving chromosomes band 161313.3. Am J Hum Genet 46:1112-1126. Wilkie AOM, Higgs DR, Rack KA, Buckle VJ, Spurr NK, FischelGhodsian N, Ceccherini I, Brown WRA, Harris PC (19911: Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16. Cell 64595-600.
