WOLF-HIRSCHHORN SYNDROME (4p-): HISTORIC, CYTOGENETIC, & MEDICAL ASPECTS Update On Advances In Knowledge John C. Carey, MD Agatino Battaglia, MD University

WOLF-HIRSCHHORN SYNDROME (4p-):WOLF-HIRSCHHORN SYNDROME (4p-):HISTORIC, CYTOGENETIC, HISTORIC, CYTOGENETIC,

& MEDICAL ASPECTS& MEDICAL ASPECTSUpdate On Advances In KnowledgeUpdate On Advances In Knowledge

John C. Carey, MDJohn C. Carey, MD Agatino Battaglia, MDAgatino Battaglia, MD University of UtahUniversity of Utah Stella Maris Institute forStella Maris Institute for

Division of Medical GeneticsDivision of Medical Genetics Child & Adolescent Child & Adolescent

Department of PediatricsDepartment of Pediatrics Neurology & Psychiatry Neurology & Psychiatry Salt Lake City, Utah Salt Lake City, Utah Pisa, Italy Pisa, Italy

Original PaperOriginal Paper• WHS is a MCA/DD disorder, first described by:

– Cooper H, Hirschhorn K. Apparent deletion of short arms of one chromosome (4 or 5) in a child with defects of midline fusion. Mammalian Chrom Nwsl 1961; 4: 14.

– Paper by Wolf et al. and Hirschhorn et al., Humangenetik, 1965

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) Syndrome

• About 200 cases reported in literature

• Very little data on the natural history Pediatrics 1999, 103: 830-836


• FREQUENCY 1/50,000 BIRTHS

• 2F : 1M SEX PREDILECTION

Exceeding 75% Greek warrior helmet appearance of the nose Microcephaly Hypertelorism Distinct Mouth Short philtrum Micrognathia Mental retardation IUGR/postnatal growth retardation Hypotonia Seizures Feeding difficulties Simple/angulated ears/pits/tags

Frequency of Main Characteristics of Wolf-Hirschhorn Syndrome

50% to 75% Distinctive EEG abnormalities Skeletal anomalies Abnormal teething Ptosis25% to 50% Heart defects Hearing defects Eye/optic nerve defect Stereotypies Cleft lip/palate Structural brain anomalies Genitourinary tract defectsBelow 25% Liver/gallbladder/gut/diaphragm anomalies

Frequency of Main Characteristics of Wolf-Hirschhorn Syndrome


• Usually there tends to be a skewing of information to the negative.

• Families being told that their child has very little chance for meaningful interaction with relatives and peers; will never walk or achieve sphincter control.


• To help delineate more accurately the natural history and to obtain better information to answer parents’ questions in a clinical setting, we collected information on >75 persons with WHS.

• Personally observed, or

• Clinical information gained from an exhaustive questionnaire sent out to the families through their national support groups in USA & Italy

(2000 – 2006).


• 2 : 1 females to males

• Age range at first observation/question. filled out: newborn to 17 years

• 20 patients followed up by us from 4 months to 22 years

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) Syndrome Birth and Family History Data Birth and Family History Data

on 60 Patientson 60 Patients

• Family histories non-contributory

• Parental ages were similar to the general population

• Almost all patients (but one) were born at term and were small for dates


• Age at diagnosis varied between 7 months gestation and 16 years

• Most patients had at least chromosome study before diagnosis was made

• Some patients with WHS are still being misdiagnosed and unrecognized


• WHS is caused by deletion of the distal

portion of the short arm of chromosome

4 (the minimal deleted segment causing

the phenotype is 4p16.3)

• Regular G-banding 60%

• HRB 15%

• FISH 25% (using a probe that includes the WHCR)

Genetics of WHSGenetics of WHSDeletions involving 4p16.3 due to different types of

rearrangements:• Cytogenetically (microscopically) visible 4p deletion (~50-

60%)

p-arm

q-arm

deletion usually larger than 4 megabases (Mb)


rearrangements:• Microdeletions (cannot be seen under the microscope)

detected by FISH using a probe for the WHS critical region (red) (~25-30%)

4

del(4)

deletion often between 2-4 Mb

WHS: cryptic deletions

Probe 190b4 (lef t) and probe 184d6 (right) deleted on one chromosome 4. Both probes identif y the WHS critical region in 4p16.3


• If needed, subtelomeric FISH screening can be performed to determine if a deletion is the result of an unbalanced translocation


• ~85% of patients have a “pure de novo deletion” (85% of paternal origin)

• ~15% patients have: “ring 4” or “4p-mosaicism”,

or “der 4” due to an unbalanced translocation

(in 2/3 mother carries the rearrangement)

• Parents of WHS should have cytogenetic analysis looking for a translocation or an inversion


• If a parent is a balanced translocation carrier, the risk to sibs of being affected with 4p monosomy (WHS) or 4p trisomy is

• The risk to the sibs of a proband is negligible if the deletion in the proband is de novo

• Prenatal testing (CVS or amniocentesis) is available if one parent is known to be a carrier of a chromosome rearrangement

AR. with WHS at

age 11 months

AR. with WHS at

age 11 months

Her boy-friend: age 8 months

Her boy-friend: age 8 months

Wolf-Hirschhorn (4p-) Syndrome:Wolf-Hirschhorn (4p-) Syndrome:Three Important ChallengesThree Important Challenges

• Feeding Difficulties

• Seizures

• Development Disability

Wolf-Hirschhorn (4p-) Syndrome Wolf-Hirschhorn (4p-) Syndrome Feeding Difficulties:Feeding Difficulties:

• Central hypotonia poor suck

• Oral facial clefts difficulty in sucking

• Poorly coordinated swallow aspirations

• G.E. reflux irritability; recurrent RTI

• G.I. malformations – rare

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) SyndromeFeeding Difficulties:Feeding Difficulties:

Health Supervision

• Sustain weight gain and health status improvement of motor abilities

• Protect the airway

• Cope with G.E. reflux

• Referral to a dysphagia team - Swallowing studies in infancy + gastrostomy tube


Feeding DifficultiesFeeding Difficulties

Treatment

• Increased caloric formulas and/or oral/nasogastric tube feeding

• Standard therapy for G.E. reflux - Nissen-Hill fundoplication

• Gastrostomy 40%

Wolf-Hirschhorn (4p-) Syndrome Wolf-Hirschhorn (4p-) Syndrome SeizuresSeizures

• Occurs in 90%

• Distinctive EEG abnormalities

Seizures In WHSSeizures In WHS• Onset between 3 to 23 months of age, with a

peak incidence at around 9 to 10 months

• Unilateral clonic/tonic, with or without secondary generalization

• Generalized tonic clonic

• On occasions lasting more than 15 minutes

• Often in clusters

Seizures In WHSSeizures In WHS

• Unilateral/generalized, clonic or tonic-clonic

status epilepticus: 60%

• Atypical absences (with a mild myoclonic component), by 1 to 5 years of age: 60%

• Seizures stopped, by 2 to 13 years of age: 33%

• Off medication: 16%

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) SyndromeSeizures/EpilepsySeizures/Epilepsy

• A candidate gene for epilepsy was the GABAA

receptor gene. Maps proximal to the WHSCR (4p12-p13).

• LETM1, a gene probably involved in Ca signaling, flanking the WHSCR (and falling within the newly proposed WHSCR-2), seems to be a good candidate for seizures.

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) Syndrome Seizures/EpilepsySeizures/Epilepsy

Treatment• Clonic/tonic-clonic seizures valproate or phenobarbital

• Atypical absences valproate ethosuccimide benzodiazepines (carbamazepine worsens)

• Clonic/tonic-clonic or absence/myoclonic status epilepticus iv. Benzodiazepines

- Dr. Battaglia

Wolf-Hirschhorn (4p-) Syndrome Wolf-Hirschhorn (4p-) Syndrome Developmental DisabilitiesDevelopmental Disabilities

• Recent studies of large samples of individuals recognition of a more complete continuum of the phenotype, pointing out a much wider clinical spectrum than previously thought (Battaglia & Carey, 2000; Battaglia et al., 2001, 2002)

Wolf-Hirschhorn(4p-) SyndromeWolf-Hirschhorn(4p-) SyndromeDevelopmental DisabilitiesDevelopmental Disabilities

Significant 65%

Moderate 25%

Mild 10%

Wolf-Hirschhorn (4P-) SyndromeWolf-Hirschhorn (4P-) SyndromeDevelopmental DisabilitiesDevelopmental Disabilities

Findings

Expressive Language:

Sounds / Words

Simple Sentences (6%)

Wolf-Hirschhorn (4P-) SyndromeWolf-Hirschhorn (4P-) SyndromeDevelopmental DisabilitiesDevelopmental Disabilities

Findings

Comprehension: Limited/Contextual

Intention to communicate: Poor/Absent in early years

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) Syndrome Developmental DisabilitiesDevelopmental Disabilities

• Sphincter control:

• Walking unsupported:

• Walking with walker:

• Help dressing/undressing:

• Doing household tasks:

age 8-14 yr (day) 10%

age 2-7 yr 27%

age 2-12 yr 18%

age 8-14 yr 18%

age 8-14 yr 18%

Findings


Findings

Follow up (20 yr): • Improvement of the motor abilities and of the disorder

of affect

• Improved adaptation to new situations; initial differentiation of the “I” processes

• Improvement of the communicative abilities and of the verbal comprehension with extension of the gesture repertoire

• Reduction of isolation and anxiety


Health Supervision

• Absent/poor speech speech evaluation

• DD psychometric evaluation

• Impaired motoric aspects motoric evaluation


Health Supervision & Treatment

• Referral to early intervention programs.

• Enrollment in an individualized rehabilitation program that covers motor aspects (including oral motor and feeding therapy), cognition, communication, and socialization.

• Appropriate school placement after full evaluation

• Planning for transition to adulthood (vocational training, living situation) to begin in adolescence.


Other Clinical Findings

• Heart anomalies: Not complex; amenable to repair

• Genitourinary: Variety of structural defects (high degree of vesicoureteric reflux) – watch for

• Eye: Coloboma, glaucoma, ptosis


Other Clinical Findings

• Dental: Altered tooth development

• Skeletal: Medically significant malformations to minor anomalies of limbs and skeleton; scoliosis

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) Syndrome Health SupervisionHealth Supervision

• Heart auscultation/EKG/echocardiography in infancy

• Renal function/renal ultrasound testing in

infancy and later

• Ophthalmology consultation in infancy

• Otolaryngological evaluation and audiological screening mandatory in infancy/childhood

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) SyndromeHealth SupervisionHealth Supervision

• Orthodontic evaluation in children/adolescents

• Club feet: early referral for evaluation/treatment

• Scoliosis/kyphosis: routine check


The right and the need of each patient with WHS to receive not just the ordinary care given to any child but also the extraordinary care necessary for coping with the problems of the del(4p) disorder.

Battaglia A, Carey JC, Wright TJBattaglia A, Carey JC, Wright TJWolf-Hirschhorn SyndromeWolf-Hirschhorn Syndrome

(Updated June 2006)(Updated June 2006)

In: GeneReviews: Genetic Disease Online Reviews at

GeneTests-GeneClinics [database online].

Copyright, University of Washington, Seattle. Available at

http://www.geneclinics.org

Battaglia, A. Battaglia, A. Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) Syndrome

in

Management of Genetic Syndromes, 2nd Edition

Cassidy SB, Allanson JE (eds)

Wiley Publishers, New York, NY, 2004.

Wolf-Hirschhorn (4p-) Syndrome:Wolf-Hirschhorn (4p-) Syndrome: Molecular Genetic PathogenesisMolecular Genetic Pathogenesis

• Over the last 10 years the WHSCR (the minimal deleted region) has been reduced to 165 kb (Wright et al., 1997). It contains two important genes of unknown function, WHSC1 and WHSC2 (Wright et al., 1997; Stec et al., 1998).

• Several candidate genes, including FGFR3, MSX1, and LETM1, fall in the flanking regions. These genes are deleted in most patients and may play a role in some aspects of the phenotype.

Wolf-Hirschhorn (4p-) SyndromeWolf-Hirschhorn (4p-) Syndrome Molecular Genetic PathogenesisMolecular Genetic Pathogenesis

• More recently, Endele et al., (2003) and Winterpacht et al., (2003) identified three novel genes (WHSC3, WHSC4, WHSC5) in the critical region


Molecular Genetic PathogenesisMolecular Genetic Pathogenesis• A new WHSCR-2, contiguous distally to the

currently defined critical region, has recently been proposed (Zollino et al., 2003)

• DFNA6, one of the>25 genes responsible for nshl has been mapped to 4p16.3snhd in 15% WHS


rearrangements:• Unbalanced translocation either new or from a parental balanced

translocation (~15-25%)

possible parental chromosomes:balanced

affected offspring: both a deletion of some gray chromosome and a duplication of some red chromosome

these rearrangements may be too small to see cytogenetically (cryptic)

Previous karyotype

Previous FISH Current array CGH results

Comparison

415

46,XY Deleted for WHS probe;

Subtelomere FISH panel: 4p deleted and replaced with 7p

CGH microarray identified same abnormalities as FISH, but also gave approximate sizes of the deletion and the duplication

CGH microarray is superior in characterizing many cryptic abnormalities

4

4p deletion 3.5-4.5 Mb

7p duplication6-7 Mb

Additional ExamplesAdditional Examples

Cytogenetic Finding CGH Microarray Result

46,XX 4p deletion and 11p duplication

46,XX 4p deletion and 8p duplication

46,XX,del(4p) 4p deletion and 11p duplication

46,XX,inv(4p) 4p deletion and 8p duplication

Limitations of CGH microarrayLimitations of CGH microarray• Will only detect the gain or loss of genomic material

that is represented on the slide

• No slide is yet clinically available that represents the entire human genome (currently can still miss very small imbalances)

• Repetitive regions of the human genome are cannot be analyzed using this technology

Chromosome ends with repetitive Chromosome ends with repetitive DNA – the acrocentricsDNA – the acrocentrics

Both the stalk and the satellite region contain only repetitive DNA and are not represented on CGH microarray slides

Previous karyotype

Previous FISH

Chromosome 4 image

CGH microarray FISH with chromosome 15p probe

636

4p deleted and replaced with unknown material

Deleted for WHS critical region

Only detected a 4p deletion

15

15

15p probe in green

4

CGH micoarray does NOT identify unbalanced translocations involving acrocentric p-arms

der(4)

2 other patients in this group also had unbalanced translocations involving an acrocentric p-arm only detected using a combination of regular cytogenetic studies (microscope) and FISH with probes specific to the acrocentric p-arms

Patients with an unbalanced translocation often Patients with an unbalanced translocation often present with an exception to some expected clinical present with an exception to some expected clinical

manifestationsmanifestations

• Microcephaly– Previous reports: present in almost all patients

(Estabrooks et. al, 1995; Zollino et. al, 2000; Buggenhout et. al, 2004)

– 3/16 did not have microcephaly– all had a cryptic deletion of 4p and duplication of 11p



• Hypospadias– Previous report: greater than 4.4 Mb deletion (Zollino et. al, 2000)

– 7/9 males in our study had hypospadias– 4/7 less than 4.4 Mb deletion and all had unbalanced

translocations• 4p deletion; 15p duplication

• 4p deletion; 14p or 22p duplication

• 4p deletion; 11p duplication (cryptic)


• Hearing loss– Previous report: greater than ~6 Mb deletion (Estabrooks et. al,

1995)

– 9/25 patients in our study had hearing loss– 3/9 less than 6 Mb deletion

2/3 had unbalanced translocations• 4p deletion; 11p duplication (cryptic)




• Heart Defects– Previous report: greater than ~6.0 Mb deletion (Zollino et. al, 2000)

– 10/25 patients in our study had heart defects– 3/10 less than 6.0 Mb deletion

2/3 unbalanced translocation• 4p deletion; 7p duplication (cryptic)


Patients with an unbalanced translocation often Patients with an unbalanced translocation often present with an exception to some expected present with an exception to some expected

clinical manifestationsclinical manifestations

ConclusionsConclusions

• CGH microarray successfully detected a deletion of 4p in each patient previously diagnosed with a 4p deletion (26/26).

• In a subset of patients (5/26), CGH microarray also detected

an additional duplication of another region not detected by a microscopic chromosome analysis plus WHS critical FISH.

• Subtelomeric FISH analysis was also able to detect these additional duplications; however, CGH microarray analysis was also able to characterize the size of the regions of deletion and duplication.

ConclusionsConclusions• CGH microarray analysis does not identify duplications of the

acrocentric p-arms

• CGH microarray should be used in conjunction with a regular karyotype analysis for optimal characterization of the genetic imbalance.

• De novo inheritance (new mutation) should not be presumed. Parental studies using the technology necessary to confirm the deletion in the child should be pursued to determine recurrence risk. – If deletion is cytogenetically visible in child, and did not require a FISH

confirmation, traditional cytogenetic analysis is sufficient for parental studies.

– If FISH was required for diagnosis of the child, parents should be studied by FISH with WHS critical region probe to rule out a cryptic balanced translocation in either parent.

ConclusionsConclusions

• Unbalanced translocations were more common than previously expected (42.3% (11/26) vs 15-25% respectively).

• Patients with an unbalanced translocation often confound some of the expected clinical manifestations.

AcknowledgementsAcknowledgements

• Primary Children’s Medical Center Foundation

• Children’s Health Research Center

• Patients with WHS, their families, and caregivers

• 4p- Support Group

• University of Utah Cytogenetics Laboratory

Documents

WOLF-HIRSCHHORN SYNDROME (4p-): HISTORIC, CYTOGENETIC, & MEDICAL ASPECTS Update On Advances In Knowledge John C. Carey, MD Agatino Battaglia, MD University