LECTURE An Approach to Work-up of Dysmorphic Patients: Clinical, Cytogenetic, and Molecular Aspects

Harold Chen

Departments of Medical Genetics and Pediatrics, University of South Alabama School of Medicine, Mobile, AL, USA

(Receivedfor publicationon December11, 1993)

Abstract. In view of recent advancement in the field of medical genetics, one approach to work-up a dysmorphic patient is to focus on how to study the patient clinically, cytogenetically, and molecularly. A clear understanding about major and minor anomalies, classification and terminologies of errors of morphogenesis, history taking, physical examination, laboratory studies including molecular cytogenetic techniques, genomic imprinting, uniparental disomy, and mosaicism is essential. Several clinical cases from my own experience are provided to illustrate my approach to work-up of dysmorphic patients. Case 1 was a newborn with multiple congenital anomalies (MCA) and a de novo dup (1) (pterg25::q12qter). Fluorescent in situ hybridization (FISH) unequivocally identified the duplicated region. Case 2 was a stillborn who had frontonasal dysplasia, arrhinencephaly, and other MCA with 46, XX, -7, +der(7), t(2;7) (q31; q36)mat. Her MCA were due to combined effect of trisomy 2q and monosomy 7q. This case helped to define the physical mapping of the critical region for a mild form of to 7q36. Case 3 had a classic de Lange phenotype with genital abnormality and sex reversal (46, XX male). Presence of SRY suggests X-Y interchange during paternal meiosis I. Case 4 was a female with primary amenorrhea, short stature, and 45, X/46, X, idic(Y)/47, X, idic(Y), idic(Y). This case demonstrates the need for molecular analyses to confirm the cytogenetic interpretations and allowed the refinement of the breakpoint in the isodicentric Y and karyotype/phenotype correlations. Case 5 & 6 were half sibs with ambiguous genitalia, minor somatic abnormalities, and dup (X) (p21.2p22.11)mat. Limited extent of the Xp duplication in these cases allows assignment of the X-linked sex-reversal (SRVX) locus to Xp21.2p22.11 . (Keio J Med 43 (2): 98-107, June 1994)

Key words: major and minor anomalies, errors of morphogenesis, mosaicism, genomic imprinting , uniparental disomy

Introduction Dysmorphology is the general term for the study of abnormal development as well as the extreme variability To work-up a dysmorphic patient usually implies to of normal physical features. One of the most difficult make a specific diagnosis, given the symptoms and signs task in dysmorphology is to determine whether a par of the patient. In view of recent advancement in the field ticular feature is pathologic or a normal variant. To be a of medical genetics, my lecture, instead, focuses on good syndromologist, we, trainers and trainees alike, how to study the patient clinically, cytogenetically, and can take note on Dr Robert Gorlin whose accomplish molecularly. A clear understanding about major and ment in the field of syndromology was honored in a minor anomalies, classification and terminologies of Festschrift issue of the American Journal of Medical errors of morphogenesis, history taking, physical exam Genetics.1 In that issue, Dr Victor McKusick commented ination, laboratory studies including molecular cyto that to make a syndromologist like Dr Gorlin , one has genetic techniques, genomic imprinting, uniparental to have a phenomenal visual memory , tremendous disomy, and mosaicism is essential. Several cases from humanity, syndrome sense, warm collegiality, strong my own clinical experience are provided to illustrate my linguistic skills, and curiosity and hard work .2 approach to work-up of dysmorphic patients.

Presented at the 839th Meeting of The Kcio Medical Society in Tokyo, October 26, 1993. Reprint requests to: Dr Harold Chen, Department of Medical Genetics, University of South Alabama, CCCB Rm 214 , Mobile, AL 36688, USA

98 Keio J Med 43 (2): 98-107, 1994 99

Major and minor anomalies condition of known cause in which there is progression and deterioration with time (e.g. Menkes disease). A About 160,000 infants will be born annually with syndrome is a given pattern of anomalies in which the anomalies and 120,000 infants will have mental retar components are known or thought to be pathogenetically dation in the United States. 3-5 Anomalies can be classi related (e.g. Down syndrome). Association is a non fied into major and minor. The major anomalies require random occurrence of multiple anomalies in 2 or more medical and surgical intervention and have serious individuals (e.g. VATER association). cosmetic impact whereas the minor anomalies are less significant and generally do not require medical inter History taking essential for work-up10 vention. Each year, 80,000 newborns, i.e. 2% of all liveborn infants, have major anomalies. Isolated anom Family history: The genetic information (e.g. name, alies, such as cleft lip/palate and , are relatively sex, date of birth, age at onset of disease, medical data, common and often multifactorial in etiology. Presence of and other essential information such as twinning and several minor anomalies increases the risk of the associ consanguinity) collected on family members in multiple ation of major defects. These structural defects are the generations can be summarized concisely in a pedigree. most common reason for genetic evaluation (71%) during This will help to determine the inheritance pattern of a the first year of life at a general genetics clinic.6 particular genetic disorder in the family. A family history of multiple spontaneous abortions or stillbirths may Classification and terminologies of errors of indicate a segregating structural chromosomal abnor morphogenesis7 mality. Ethnic background may be important in certain genetic disorders (e.g. sickle cell anemia in blacks; Tay Malformation, disruptions, deformations, dysplasias: - Sachs disease in Ashkenazic Jews). Parental ages are Anomalies are commonly classified into malformations, important: an advanced maternal age is associated with disruptions, deformation, and dysplasia, depending on an increased risk of having a child with a chromosomal the presumed pathogenesis.8 Opitz in 19829 clarified anomaly (e.g. trisomy 21 syndrome); advanced paternal and expanded the concepts and terminology used in age may suggest an autosomal dominant new mutation the classification so that phenotypes can be analyzed (e.g. achondroplasia). correctly, thereby providing the basis for accurate diag nosis and proper counseling. Malformations are struc Pregnancy and birth history: Maternal risk factors tural defects caused by intrinsic or extrinsic insults during (e.g. infections, drug administration, radiation, malnu embryogenesis (e.g. holoprosencephaly). Most malfor trition, anoxia, trauma, diabetes, and other compli mations are developmental field defects. A developmental cations) during pregnancy should be recorded. It is also field is a region or part of an embryo that responds as a important to note fetal activity, fetal size, fetal presen coordinated unit to embryonic interaction and results in tation, the amount and appearance of amniotic fluid, complex or multiple anatomic structures. Disruptions gestational age, Apgar scores, and a description of the arise after formation of structures by destructive pro placenta, including the fetal membranes and the number cess (e.g. amniotic deformation-adhesion-mutilation of cord vessels. complex). A deformation usually arise in late gestation and is caused by mechanical force on various structures Medical information: Adequate and essential infor (e.g. talipes equinovarus deformity or Potter syndrome mation is needed for accurate diagnosis of a genetic caused by oligohydramnios). A dysplasia is an abnormal disorder. Personal medical records, including biopsy organization of cells into tissues and its morphologic slides, autopsy reports, and results of special studies results (e.g. hemangiomas). such as radiographs, CT scans, MRI, and biochemical , cytogenetic, and molecular studies should be requested Sequence, disease, syndrome, association: A sequence and reviewed if necessary. Permission to contact relatives is a pattern of multiple anomalies derived from a single and to acquire photographs may need to be obtained . prior anomaly or mechanical factor. A sequence may be a malformation sequence (e.g. meningomyelocele Physical examination produces lower limb paralysis, muscle wasting, clubfeet, incontinence, urinary tract infections, and other abnor Major anomalies are obvious on physical examination; malities), a disruption sequence (ADAM sequence minor anomalies, however , require careful observation, caused by early amnion rupture), a deformity sequence including measurements and comparisons to normal (e.g. Potter sequence caused initiallyby oligohydramnios), growth charts, since they often provide important clues and a dysplasia sequence (renal, pulmonary, CNS, and to diagnosis. When there are three or more minor anom cardiac sequences in tuberous sclerosis). A disease is a alies, one should look for occult major anomalies . 100 Chen H: Work-up of Dysmorphic Patients

Physical examinations should include anthropometric ing ambiguous or hypoplastic genitalia and failure of measurements (height, weight, head circumference, sexual maturation at puberty. (4) Unexplained abnormali other pertinent measurements of features that appear ties of growth. (5) Features of mosaicism (hypomelanotic unusual or abnormal), and an estimate of the develop spots, streaky pigmentary disturbance, asymmetry). (6) mental status (e.g. speech delay, mental retardation). Spontaneously aborted embryos, unexplained stillborn Repeat observations over a period of years may ulti fetuses, and hydropic placentas. (7) Family members of mately lead to a diagnosis not initially obvious or revision an individual with an inherited chromosome duplication, of a previously incorrect diagnosis.11 Comparison of deletion or other rearrangement. minor features, particularly facial characteristics, with those of the parents and siblings often permits separation Fluorescence in situ hybridization (FISH)13,14 of family traits from those that may be of diagnostic importance. FISH has become an important tool for analysis of the Natural histories of various syndromes are important number, size, and location of specific DNA sequences. to keep in mind. For examples, in the fragile X syndrome, Chromosome specific probes are of 3 types; chromosome one expects large size at birth; developmental delay, specific repeat-sequence (centromere) probes, whole especially in speech, during the early childhood; and chromosome "painting" probes, and locus-specific probes. facial characteristics and macroorchidism in the post The ability to detect and characterize numerical and pubertal years.11 Infants with 45,X Turner syndrome structural aberrations in metaphase spreads and in inter have lymphedema, excess nuchal skin, and coarctation phase nuclei has substantial clinical value. of the aorta at birth; stunted growth during childhood; and failure to mature sexually at adolescence.12 Molecular cytogenetics

Laboratory studies The first successful mapping of a mendelian disorder by chromosome rearrangements was mapping of the In most cases, laboratory studies include blood, urine, Duchenne muscular dystrophy locus to Xp21.15 Other and biopsied samples. Fibroblast and lymphoblastoid chromosome rearrangements (e.g. deletions, translo cell lines are important for the studies of certain con cations) not only helped the mapping of certain disease ditions, especially chromosomal and metabolic disorders. genes but also contributed to rapid isolation of disease genes by positional cloning strategies.16 Imaging techniques Mosaicism The radiographic examination is probably the most useful means of studying skeletal dysplasia. The short Mosaicism denotes the presence of at least two cell stature can be predominantly in the trunk or in the lines differing in genotype or karyotype, in a single limbs. Diagnosis of the short limb variety depends on individual or tissue, derived from a single zygote. Current the segment of the long bone most severely affected (e.g. evidence suggests that mosaicism is a common, possibly rhizomelic, mesomelic, acromelic, and acromesomelic universal phenomenon.17 Functional mosaicism has been shortenings). Diagnosis can also be based on the affected produced regularly by X-inactivation and tissue differ anatomical part of the skeleton that can be detected entiation. Mosaicism can be found both in somatic cells radiologically (e.g. epiphyseal, metaphyseal, diaphyseal, and germ cells. For instance, a parent with a patchy or spondyloepiphyseal, spondylometaphyseal, and spon segmental neurofibromatosis (appears to be somatic dyloepimetaphyseal dysplasias). mosaics) with fully affected children would suggest that the parent has both germ line and somatic mosaicism.18 Cytogenetic studies Patchy or streaky pigment indicates gene or chromo some mosaicism. For examples, patients with McCune Chromosome analysis plays an important role in - Albright syndrome, which is characterized by sexual evaluating dysmorphic patients. Indications for cytogen precocity, osteolytic lesions, and hyperpigmentation, are etic studies include: (1) Clinical features suggestive of mosaic for genes (mutations of a subgroup of the G1 recognizable chromosomal syndromes. (2) Patients with protein) in the affected areas.19 Patients with hypo idiopathic multiple congenital anomalies and mental melanosis of Ito,20 which is characterized clinically by retardation (MCA/MR) syndrome, especially when patchy depigmentation and CNS deterioration, and patients have an unusual facial appearance, microcephaly, many patients who have asymmetric growth 21 have been prenatal and postnatal growth deficiency, or several found to be chromosomally mosaic. minor anomalies, and there is a lack of familial resem blance. (3) Abnormality of sexual development, includ Kcio J Med 43 (2): 98-107, 1994 101

Nontraditional inheritance (genomic imprinting, uniparental disomy)22-25

Mendelian genetics implies that identical genes inherited from each parent have an equal effect on the development of their offspring, i.e. the parental source of the genetic information does not influence gene expression. During the last decade evidence has accumulated in mammalian genetics that certain genetic phenomena cannot be explained by traditional mendelian concepts. Genomic (parental) imprinting and uniparental disomy, two important phenomena and interrelated concepts, have been described recently.

Genomic imprinting: Genomic imprinting is defined Fig 1 Southern blots show the parental origin of the deletion of as the differential influence of genetic material, being Angelman syndrtome (AS) and Prader-Willi syndrome (PWS). The transferred to the embryo from the mother or father, maternal allele (3.8kb) was absent in the AS patient while the paternal allele (also 3.8kb) was absent in the PWS patient. on development of the embryo. Deficient or excessive maternal or paternal influence (imprinting) modifies the phenotype of the placenta or embryo. Genomic imprinting on the placenta is dominated by paternal hands (puppet-like motion), severe mental retardation chromosomal influence; genomic imprinting on the especially absence of speech, microbrachycephaly, and embryo by maternal chromosomal influence. When uncontrollable seizures. Deletion of the same chromo there is only paternally derived chromosome material, some region 15q11-13 also occurred in AS patients.28,29 there is relatively normal development of the placenta The parental origin of the deletion distinguishes with poor development of the embryonic structures; PWS and AS; paternal deletion in the former 30,31 and when there is only maternally derived chromosome maternal deletion in the latter. 32,33Fig. 125 (modified material, there is reasonable development of the embryo from Driscoll, 1993) shows the parental origin of the with poor placental development. The following two deletion in the AS and PWS using Southern blotting clinical examples reflect these phenomena: hydatidiform technique, digested with the restriction enzyme TagI and moles, a placental tumor, have both sets of paternally hybridized with the probe 189-1 from the 15q11-13 derived chromosomes while ovarian tumors, which region. 31 The maternal allele (3.8kb) was absent in the consist of embryonic tissue, have only maternally derived AS patient and the paternal allele (3.8kb) was absent in chromosomes. the PWS patient. A parent-of-origin DNA methylation In humans, examples of genomic imprinting26 are: (D15S9 and D15S63 loci), DNA replication asynchrony Angelman and Prader-Willi syndrome (15q11-13), between maternal and paternal derived chromosomes, fragile X syndrome (Xq27.3), Huntington disease (distal DNA binding proteins and chromatin structure have all 4p), myotonic dystrophy (19q13.3), teratomas and been implicated in the imprinting process.34 hydatidiform moles (all chromosomes), and Wilms tumor (1lp13-15). The most compelling evidence that genomic Uniparental disomy (UPD): A significant number of imprinting exists in humans and plays an etiological role non-deletion cases of AS and PWS patients exist. Nicholls in causing anomalies came from the Angelman and et al31 demonstrated two maternal copies of chromosome Prader-Willi syndrome. Both syndromes have strikingly 15 and no paternal copy as a mechanism in causation of different neurobehavioural characteristics but share a the PWS. This phenomenon is called uniparental disomy. common chromosomal deletion in 15q11-q13. UPD originates when 2 copies of a particular chromo Prader-Willi syndrome (PWS) is characterized by some come from one parent and none from the other profound obesity secondary to hyperphagia, hypog parent. Uniparental isodisomy occurs where 2 copies of onadism, mild to moderate mental retardation, profound exactly the same chromosome originated from one hypotonia in infancy, small hands and feet, and picking parent; uniparental heterodisomy occurs where both at skin sores. Using high resolution chromosome analysis, homologs of a chromosome pair originated from one Ledbetter et al27 demonstrated for the first time a small parent. Later, UPD was also demonstrated to be a deletion at 15q11-13 in PWS patients. mechanism in the AS.35 Dependent upon which parental Angelman syndrome (AS) is characterized by unu chromosome region (15q11-13) is missing , there is sually happy disposition, inappropriate laughter, affec a significant difference in the phenotype . Lack of a tionate behavior, ataxic, unsteady gait with upraised paternal region, either by deletion or UPD, leads to 102 Chen H: Work-up of Dysmorphic Patients

Fig 3 Hypothetical pedigree for paternal imprinting is shown. No phenotypic expression when inherited from the father.

Fig 2 Molecular classes of AS and PWS patients are shown. P represents paternal chromosome 15 (shown in solid bar); M represents maternal chromosome 15 (shown in blank bar).

PWS; lack of a maternal region by the same mechanisms leads to AS. Fig 4 Hypothetical pedigree for maternal imprinting is shown. No Molecular classes of AS and PWS patients are given phenotypic expression when inherited from the mother. in the Fig 2.25Molecular deletions are seen with similar frequencies in both conditions (72% AS vs 74% PWS). The incidence of UPD is frequent in PWS (29%) but typic expression when inherited from the father; the infrequent in AS (5%). The biparental class of AS term maternal imprinting is used to imply that there (23%) (neither deletion nor UPD) is probably due will be no phenotypic expression when inherited from to mutation in a single maternal gene. The gene(s) the mother. responsible for AS and PWS undoubtedly are in close proximity but must be separate loci. Case Illustrations Possible mechanisms for the origin of UPD are; 1) simultaneous nondisjunction in maternal and paternal Case 1 gametogenesis (one parent contributes two chromosome 15's and the other parent contributes none), 2) non A newborn with multiple congenital anomalies (Fig 5) disjunction in one parent with two chromosome 15's died shortly after birth. Clinical features were charac leading initially to a trisomic state with subsequent terized by micro/brachycephaly, hydrocephalus, small reduction by selection to a disomic state. temporal lobes, cardiac defects consisting of patent ductus arteriosus and foramen ovale, pulmonary hypo

Patterns of inheritance of imprinted genes23 plasia, eventration of left hemidiaphragm, cryptorchidism, sacral dimple, flexion contractures of fingers/, and It is important to re-examine pedigrees in known talipes equinovarus deformities. Chromosome analysis disorders for possible imprinting effects since imprinting revealed de novo dir dup(1) (pter•¨q25::q12•¨qter) appears to be a common phenomenon in humans. There (Fig 6). may be differences in phenotypic expression depending Most cases of dup(1q) involve distal 1q and usu on who is the parent transmitting the gene. If a gene is ally associated with monosomy of other chromosomal imprinted, it is "silenced" or "turned off" when it has segments. Pure proximal dup(1q) are extremely rare. been inherited from one particular parent, mother FISH technique is essential to accurately define the or father. duplication segment of this case although C-banding

Hypothetical pedigrees are shown for paternal (Fig 3) pattern did suggest possibble duplication of chromosome and maternal (Fig 4) imprinting. The term paternal 1. This is the first case with unequivocal identification imprinting is used to imply that there will be no pheno- of proximal duplication of 1q, using a chromosome Keio J Med 43 (2): 98-107, 1994 103

Fig 6 Idiogram (a) and partial G-banded (b) and C-banded (c) karyotypes illustrating the normal (left) and the duplicated (right) chromosome 1.

Fig 5 Frontal (a) and lateral (b) view of the patient. & maternal grandmother both had balanced t(2;7) (q31;q36). The stillborn (Fig 9) had intrauterine growth 1 painting probe (Fig 7).36 The FISH technology is, retardation, frontonasal dysplasia, arrhinencephaly therefore, essential for accurate karyotypic diagnosis and other CNS abnormalities, pulmonary hypoplasia, and should help in further karyotypic-phenotypic cor cartilaginous deficiency of trachea and bronchus, severe relation of partial 1q trisomy. tetralogy of Fallot, gastrointestinal tract anomalies (Meckel diverticulum, colon malrotation), genitourinary Case 2 tract anomalies (renal and ureteral aplasia, bladder hypoplasia, imperforate anus), and flexion contractures Fetal blood sampling was performed because of of joints with webbings. abnormal ultrasound findings (oligohydramnios and fetal This is an important case. Frontonasal dysplasia has ocular ). The karyotype revealed 46, XX, not been reported to be associated with chromosome - 7, +der(7), t(2;7)(q31;q36)mat (Fig 8). The mother anomaly. Multiple congenital anomalies of this patient

Fig 13 FISH analysis . The sites are separated by the combined length of most of the short of Y . A chromosome spread shows 2 idic(Y) .

Fig 7 Partial chromosome spread countcrstaincd with DAPI (a) and hybridized with the chromosome 1 painting probe labeled with Texas red (b). 104 Chen H: Work-up of Dysmorphic Patients

Case 3

An infant (Fig 10) was referred for evaluation because of small for gestational age, hirsutism, microcephaly, synophyrys, long/curled eyelashes, small nose, a carp like upper lip with a receding chin, low set ears, micro penis with hypospadias, and (). The diagnosis suggests Cornelia de Lange syndrome. Genital abnormality in the de Lange syndrome is quite rare. This prompted chromosome analysis which revealed 46,XX. Since the cytogenetic finding (46,XX) did not correspond to the phenotypic sex (male), further mol ecular studies were performed. PCR analysis using Y97 alphoid repeat sequence revealed no presence of Y centromeric repeat sequence. This implies that there is no free Y chromosome present Fig 8 Partial karyotypes of the patient, mother and maternal grandm in the patient. PCR analysis using SRY primers revealed other and an idiogram illustrating t(2:7)(q31;q36). a 609by fragment (Fig 11), indicating the presence of SRY. Speculation is that the tip of one of the X chromo some contains SRY, which originated from X-Y inter may be due to partial trisomy 2q, but effect of the change during meiosis I of the father. This could be monosomy of 7q cannot be overlooked. Recently, the demonstrated by FISH with SRY probe to hybridize the gene for a form of holoprosencephaly has been suggested tip of the short of an X chromosomes. at 7q36. This case further supports the physical mapping of the critical region for a mild form of holoprosencephaly Case 4 to 7q36. We have also defined the minimal critical region of holoprosencephaly to be in 7q36 between DNA An 18 year old female was evaluated for primary markers D7S92 and D7S392.37 amenorrhea and short stature. There was hypoplastic labia majora with clitoromegaly. Cytogenetic studies

Fig 9 Whole view of the patient showing characteristic craniofacial features along with flexed elbows and wrists, flexed Fig 10 (a) Frontal view of the patient showing characteristic craniofacial features and ectrodactyly of fingers with intcrphalangeal webbings, Cornelia de Lange syndrome. (b) Genitalia showing micropenis with hypospadias and cryptorchidism . fixed , extended knees, and rotational deformities of the ankles and feet. Keio J Med 43 (2): 98-107, 1994 105

Fig 11 PCR analysis using SRY primers revealed a 609 by fragment in lanes 2 and 3; lanes 4 and 5 are male controls; lanes 6 and 7 are female Fig 12 Q-banded metaphase showing the idic(Y) with brightly fluor controls. Lane 1 is a standard DNA markers. escent bands (Yqh region) at each end of the chromosome.

were carried out. Three cell lines were present: 45, X/ 46, X, idic(Y)/47, X, idic(Y), idic(Y). The isodicentric Y chromosome was described as idic(Y) (qterp11; p11qter) (Fig 12). The results of cytogenetic analysis were variable in different tissues (blood, skin, right and left gonad). Internal genital organs were characterized by a rudimentary uterus, left vestigial Wolffian structures in the left streak gonad, left fallopian tube, and right dysgerminoma. A total of 38 cases of dic(Y) with breakpoint in the Yp have been reported in the literature; 38 ambiguous external Fig 14 Amplified SRY fragment (609bp) was present in blood (4), skin (lane 5) and right gonad (lane 6) but absent in the left gonad (lane genitalia with streak gonads, undescended testes, and 7). Lane 1, standard marker PBR322, MSPI digest; lane 2, male ovotestes (31.6%), female external genitalia (44.7%) control; lane 3, female control: lane 8, proband's mother; lane 9, is with some degree of gonadal dysgenesis, and phenotypic proband's father. male (mostly infertility) (23.7%). Short stature, features of Turner syndrome, and gonadoblastoma were reported in 63%, 26%, and 18% respectively.

To further define the idic(Y), FISH was performed. the Yp were duplicated. ZFY and SRY which map near The binding sites of biotin-labeled ƒ¿-satellite probe of Y the pseudoautosomal boundary are both present, placing are seen at the 2 centromeric regions of the dicentric Y. the breakpoint at Yp11.32. PCR analysis confirmed the The sites are separated by the combined lengths of most cytogenetic results of tissue distribution of the idic(Y) . of the 2 short arms of the Y chromosome (Fig 13). The probe is detected with fluorescein and chromosomes Cases 5 and 6 are counterstained with propidium iodide. FISH also confirmed the presence of the 2 idic(Y). Phenotypically normal mother was found to have The following molecular studies were performed. 46, X, dup(X) (p21.2•¨p22.11) (Fig 15). Her two children

PCR analysis using ZFY primers amplified a Y-linked (half sibs) have 46, X, dup(X) (p21.2p22.11)mat, Y zinc finger fragment (340bp) in the proband. PCR analy (Fig 15). Both children have ambiguous genitalia sis was also performed using SRY primers. Amplified (Fig 16) and gonads resembling epididymis without

SRY fragment (609pb) was absent in left gonad but germ cells and no mullerian derivatives. The older present in right gonad, skin, and blood (Fig 14). Results child had mild developmental delay; the younger child of the molecular analysis were compatible with the had . cytogenetic analyses. Female differentiation of XY embryos may be caused FISH analysis confirmed the presence of the third cell by mutation in at least the following genes; 1) SRY , the line with 2 dic(Y). The entire long arm and most of Y-linked testis-determining gene; 2) SRA1 (17q24 .3- 106 Chen H: Work-up of Dysmorphic Patients

q25.1), the autosomal sex reversal gene associated with campomelic dysplasia;39 3) an X-linked gene, indicated by certain familial cases of XY gonadal dysgenesis and in sex-reversed XY females with duplications in Xp including this family. We have now mapped the X-linked gene, SRVX, based on our study of two families with XP21 duplication;40the first family41in which dup(Xp2lpter) was found in a XY male with severely retardation and MCA (Gardner-Silengo-Wachtel syndrome) and her similarly affected fetal sibling; the second family42,43 in which the duplication involved Xp21 2p22.11 in sex reversed maternal half siblings with only minor somatic abnormalities. The limited extent of the Xp duplication in the second family allows assignment of the SRVX locus to Xp21.2p22.11, spanning about 6 or 7 megabases distal to the OTC locus and including DMD. When the duplication does not include Xp21.3, the genitalia are those of a normal male, 44,45suggesting that the region containing SRVX may be further localized to Xp21.1p21.3.46

Acknowledgements: The author is indebted to Dr Gen Nishimura and Dr Nobutake Matsuo for the opportunity of presenting this special lecture at the 839th Meeting of the Keio Medical Society in Tokyo, October 26, 1993.

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